Sterile chromatography resin and use thereof in manufacturing processes

ABSTRACT

Provided herein are methods of reducing bioburden of (e.g., sterilizing) a chromatography resin that include exposing a container including a composition including a chromatography resin and at least one antioxidant agent and/or chelator to a dose of gamma-irradiation sufficient to reduce the bioburden of the container and the chromatography resin, where the at least one antioxidant agent and/or chelator are present in an amount sufficient to ameliorate the loss of binding capacity of the chromatography resin after/upon exposure to the dose of gamma-irradiation. Also provided are reduced bioburden chromatography columns including the reduced bioburden chromatography resin, compositions including a chromatography resin and at least one chelator and/or antioxidant agent, methods of performing reduced bioburden column chromatography using one of these reduced bioburden chromatography columns, and integrated, closed, and continuous processes for reduced bioburden manufacturing of a purified recombinant protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/598,401, filed on Jan. 16, 2015, which claimspriority to U.S. Provisional Patent Application Ser. No. 61/928,929,filed on Jan. 17, 2014, and U.S. Provisional Patent Application Ser. No.62/001,498, filed on May 21, 2014, which are incorporated by referencein their entirety.

TECHNICAL FIELD

This invention relates to methods of biotechnology and thebiomanufacturing of recombinant proteins.

BACKGROUND

Mammalian cells including a nucleic acid that encodes a recombinantprotein are often used to produce therapeutically or commerciallyimportant proteins. In the current environment of diverse productpipelines, biotechnology companies are increasingly driven to developinnovative solutions for highly flexible and cost-effectivemanufacturing of therapeutic protein drug substances. One of thestrategies for efficiently isolating recombinant proteins is throughprocesses that include continuous chromatography (e.g., using a closedsystem). One known limitation of continuous chromatography is thepresence of contaminating agents in the system (e.g., increasedbioburden), which results in a contaminated product, a reduction in theproduction yield, and a decrease in the flow-rate (or increase in thepressure) in the system. For example, the increased bioburden within asystem can result in the complete shut down of the system.

SUMMARY

The present invention is based, at least in part, on the discovery thatgamma-irradiation of chromatography resin reduces the binding capacityof the chromatography resin. In view of this discovery, provided hereinare methods of reducing bioburden of a chromatography resin that includeexposing a container including a composition including a chromatographyresin and at least one antioxidant agent and/or chelator to a dose ofgamma-irradiation sufficient to reduce the bioburden of the containerand the chromatography resin, where the at least one antioxidant agentand/or chelator are present in an amount sufficient to ameliorate theloss of binding capacity of the chromatography resin after/upon exposureto the dose of gamma-irradiation. Also provided are methods of reducingbioburden of a chromatography resin that include exposing a containerincluding a composition including a chromatography resin (and optionallyat least one antioxidant agent and/or chelator in an amount sufficientto ameliorate loss of binding capacity of the chromatography resinafter/upon exposure to gamma-irradiation) to gamma-irradiation at a rateof between about 0.1 kGy/hour to about 6 kGy hour and/or at atemperature between about 4° C. to about 25° C., for a dose ofgamma-irradiation sufficient to reduce the bioburden of the containerand the chromatography resin. Also provided are reduced bioburdenchromatography columns containing a reduced bioburden chromatographyresin prepared by any of the methods described herein, compositionsincluding a chromatography resin and at least one chelator and/orantioxidant agent, methods of performing reduced bioburden columnchromatography using at least one of these reduced bioburdenchromatography columns, and integrated, closed or substantially closed,and continuous processes for reduced bioburden manufacturing of apurified recombinant protein that include the use of at least one ofthese reduced bioburden chromatography columns. Any of thechromatography resins produced by any of the methods described herein,any of the packed chromatography columns produced by any of the methodsdescribed herein, any of the methods of performing columnchromatography, and any of the processes described herein can besterile, absolutely sterile, aseptic, or reduced bioburden. Any of thechromatography resins produced by any of the methods described herein,any of the chromatography columns produced by any of the methodsdescribed herein, and any of the processes described herein can beaseptic and sterile, absolutely sterile, aseptic, or reduced bioburden.

Provided herein are methods of reducing bioburden of a chromatographyresin that include: exposing a container including a compositionincluding a chromatography resin and at least one antioxidant agentand/or chelator to a dose of gamma-irradiation sufficient to reduce thebioburden of the container and the chromatography resin, where the atleast one antioxidant agent and/or chelator is present in an amountsufficient to ameliorate the loss of binding capacity of thechromatography resin after exposure to the dose of gamma-irradiation.Some embodiments of these methods further include, prior to exposing,disposing the composition into the container. In some examples, theirradiation is performed at a rate of between about 0.1 kGy/hour toabout 6 kGy hour and/or at a temperature between about 4° C. to about25° C. In some examples of any of these methods, the container is astorage vessel, a chromatography column, or a packed chromatographycolumn. In some examples of any of the methods described herein, thecomposition is a slurry of sedimented chromatography resin in a liquidincluding the at least one antioxidant agent and/or chelator. In someexamples, the liquid comprises: (i) between 75 mM and about 125 mM(e.g., between 80 mM and about 120 mM, between about 85 mM and about 115mM, between about 90 mM and about 110 mM, or between about 95 mM andabout 105 mM) mannitol; (ii) between 75 mM and about 125 mM (e.g.,between about 80 mM and about 120 mM, between about 85 mM and about 115mM, between about 90 mM and about 110 mM, or between about 95 mM andabout 105 mM) methionine (or alternatively cysteine or glutathione);(iii) between 75 mM and about 125 mM (e.g., between about 80 mM andabout 120 mM, between about 85 mM and about 115 mM, between about 90 mMand about 110 mM, or between about 95 mM and about 105 mM) sodiumascorbate; (iv) between 75 mM and about 125 mM (e.g., between about 80mM and about 120 mM, between about 85 mM and about 115 mM, between about90 mM and about 110 mM, or between about 95 mM and about 105 mM)histidine; (v) between 30 mM and about 70 mM (e.g., between about 35 mMand about 65 mM, between about 40 mM and about 60 mM, or between about45 mM and about 55 mM) methionine (or alternatively cysteine orglutathione) and between about 30 mM and about 70 mM (e.g., betweenabout 35 mM and about 65 mM, between about 40 mM and about 60 mM, orbetween about 45 mM and about 55 mM) histidine; (vi) between about 10 mMand about 50 mM (e.g., between about 15 mM and about 45 mM, betweenabout 20 mM and about 40 mM, or between about 25 mM and about 35 mM)methionine (or alternatively cysteine or glutathione), between about 10mM and about 50 mM (e.g., between about 15 mM and about 45 mM, betweenabout 20 mM and about 40 mM, or between about 25 mM and about 35 mM)histidine, and between about 10 mM and about 50 mM (e.g., between about15 mM and about 45 mM, between about 20 mM and about 40 mM, or betweenabout 25 mM and about 35 mM) sodium ascorbate; or (vii) between about 5mM to about 45 mM (e.g., between about 10 mM and about 40 mM, betweenabout 15 mM and about 35 mM, or between about 20 mM and about 30 mM)sodium ascorbate, between about 5 mM and about 45 mM (e.g., betweenabout 10 mM and about 40 mM, between about 15 mM and about 35 mM, orbetween about 20 mM and about 30 mM) methionine (or alternativelycysteine or glutathione), between about 5 mM and about 45 mM (e.g.,between about 10 mM and about 40 mM, between about 15 mM and about 35mM, or between about 20 mM and about 30 mM) mannitol, and between about5 mM and about 45 mM (e.g., between about 10 mM and about 40 mM, betweenabout 15 mM and about 35 mM, or between about 20 mM and about 30 mM)histidine. In some examples, the liquid is a buffered solution (e.g., aphosphate buffered solution, e.g., a sodium phosphate buffered solution,such as 50 mM sodium phosphate, pH 6.0).

In some examples of any of the methods described herein, the compositionis a solid mixture. In some embodiments of any of the methods describedherein, the composition includes at least one antioxidant agent selectedfrom the group of: reduced glutathione, reduced thioredoxin, reducedcysteine, a carotenoid, melatonin, lycopene, tocopherol, reducedubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, a flavonoid, aphenolpropanoid acid, lidocaine, naringenin, fullerene, glucose,mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, anddimethylmethoxy chromanol. In some embodiments of any of the methodsdescribed herein, the composition includes at least one (one, two,three, or four) antioxidant agent selected from mannitol, sodiumascorbate, methionine (or alternatively cysteine or glutathione), andhistidine. In some embodiments, the composition includes mannitol,sodium ascorbate, methionine (or alternatively cysteine or glutathione),and histidine. In some embodiments of any of the methods describedherein, the composition includes at least one chelator selected from thegroup of: ethylenediaminetetraacetic acid (EDTA),2,3-dimercapto-1-propanesulfonic acid sodium (DMPS), dimercaptosuccinicacid (DMSA), metallothionin, and desferroxamine.

In some examples of any of the methods described herein, the compositionincludes at least one chromatography resin selected from the group of:anionic exchange chromatography resin, cationic exchange chromatographyresin, affinity or pseudo-affinity chromatography resin, hydrophobicinteraction chromatography resin, and size exclusion chromatographyresin (or any combination thereof). In some examples of any of themethods described herein, the chromatography resin can be multi-modal(e.g., bi-modal) chromatography resin (e.g., a chromatography resin thathas anionic exchange and hydrophobic interaction groups, or achromatography resin that has both cation exchange and hydrophobicinteraction groups). In some examples of any of the methods describedherein, the composition includes affinity chromatography resin includinga protein ligand (e.g., protein A or protein G). In some examples of anyof the methods described herein, the composition includes an anionicexchange chromatography resin (e.g., an anionic exchange chromatographyresin including N-benzyl-N-methyl-ethanolamine groups).

In some examples of any of the methods described herein, the dose isbetween about 2 kGy to about 45 kGy (e.g., between about 20 kGy to about30 kGy, between about 23 kGy and about 27 kGy, between about 2 kGy toabout 40 kGy, between about 2 kGy and about 35 kGy, between about 2 kGyand about 30 kGy, between about 2 kGy and about 25 kGy, between about 10kGy and about 45 kGy, between about 10 kGy and about 40 kGy, betweenabout 10 kGy and about 35 kGy, between about 10 kGy and about 30 kGy, orbetween about 10 kGy and about 25 kGy). In some embodiments of any ofthe methods described herein, the exposing is performed at a temperaturebetween about −25° C. and about 0° C., inclusive, or between about 0° C.and about 25° C., inclusive (e.g., between about 4° C. and about 25°C.).

Also provided is a reduced bioburden chromatography resin produced byany of the methods described herein. In some examples of any of thereduced bioburden bioburden chromatography resins provided herein have asterility assurance level (SAL) of between about 1×10⁻⁸ to about 1×10⁻⁵(e.g., between about 1×10⁻⁷ to about 1×10⁻⁶). For example, achromagraphy resin produced by any of the methods described herein canhave reduced bioburden, is sterile, is aseptic, or is absolutelysterile. In some embodiments, a chromatography resin produced by any ofthe methods described herein can be aseptic and have reduced bioburden,be sterile, or be absolutely sterile. Some examples of any of thereduced bioburden chromatography resins provided herein include at leastone resin selected from the group of: anionic exchange chromatographyresin, cationic exchange chromatography resin, affinity orpseudo-affinity chromatography resin, hydrophobic interactionchromatography resin, and size exclusion chromatography resin. In someembodiments, the reduced bioburden chromatography resin provided hereinincludes affinity chromatography resin including a protein ligand (e.g.,protein A or protein G). In some embodiments, the reduced bioburdenchromatography resin provided herein includes anionic exchangechromatography resin (e.g., an anionic exchange chromatography resinincluding N-benzyl-N-methyl-ethanolamine groups). In some embodiments,the reduced bioburden chromatography resin provided herein ismulti-modal chromatography resin (e.g., a bimodal chromatography resin).

Also provided are methods of making a reduced bioburden packedchromatography column that include: providing any of the reducedbioburden chromatography resins produced by any of the methods describedherein; and packing the chromatography resin into a reduced bioburdencolumn in an aseptic environment. Also provided are reduced bioburdenpacked chromatography columns that are produced by gamma-irradiating acomposition including a packed chromatography resin and at least oneantioxidant agent and/or chelator (e.g., any of the exemplarycompositions provided herein) included within a chromatography column.Any of the reduced bioburden packed chromatography columns providedherein can have a sterility assurance level (SAL) of between about1×10⁻⁸ to about 1×10⁻⁵ (e.g., between about 1×10⁻⁷ to about 1×10⁻⁶). Anyof the chromatography columns produced by any of the methods describedherein can have reduced bioburden, be sterile, be absolutely sterile, orbe aseptic. For example, any of the chromatography columns produced byany of the methods described herein can be aseptic and can have reducedbioburden, be sterile, or be absolutely sterile.

In some embodiments of any of the reduced packed chromatography columnsprovided herein, the resin in the packed column includes at least oneresin selected from the group of: anionic exchange chromatography resin,cationic exchange chromatography resin, affinity chromatography resin,hydrophobic interaction chromatography resin, and size exclusionchromatography resin. In some embodiments of any of the reducedbioburden packed chromatography columns provided herein, the resinincludes affinity chromatography resin including a protein ligand (e.g.,protein A or protein G). In some examples of any of the reducedbioburden packed chromatography columns provided herein, the resinincludes anionic exchange chromatography resin (e.g., an anionicexchange chromatography resin includes N-benzyl-N-methyl-ethanolaminegroups).

Also provided are compositions including a chromatography resin and atleast one antioxidant agent and/or chelator, where the at least oneantioxidant agent and/or chelator is present in an amount sufficient toameliorate the loss of binding capacity of the chromatography resin upontreatment with a dose of gamma-irradiation sufficient to reducebioburden of the composition. In some examples of any of thecompositions provided herein, the composition is a slurry of sedimentedchromatography resin in a liquid including the at least one antioxidantagent and/or chelator. In some examples, the liquid comprises: (i)between 75 mM and about 125 mM (e.g., between 80 mM and about 120 mM,between about 85 mM and about 115 mM, between about 90 mM and about 110mM, or between about 95 mM and about 105 mM) mannitol; (ii) between 75mM and about 125 mM (e.g., between about 80 mM and about 120 mM, betweenabout 85 mM and about 115 mM, between about 90 mM and about 110 mM, orbetween about 95 mM and about 105 mM) methionine (or alternativelycysteine or glutathione); (iii) between 75 mM and about 125 mM (e.g.,between about 80 mM and about 120 mM, between about 85 mM and about 115mM, between about 90 mM and about 110 mM, or between about 95 mM andabout 105 mM) sodium ascorbate; (iv) between 75 mM and about 125 mM(e.g., between about 80 mM and about 120 mM, between about 85 mM andabout 115 mM, between about 90 mM and about 110 mM, or between about 95mM and about 105 mM) histidine; (v) between 30 mM and about 70 mM (e.g.,between about 35 mM and about 65 mM, between about 40 mM and about 60mM, or between about 45 mM and about 55 mM) methionine (or alternativelycysteine or glutathione) and between about 30 mM and about 70 mM (e.g.,between about 35 mM and about 65 mM, between about 40 mM and about 60mM, or between about 45 mM and about 55 mM) histidine; (vi) betweenabout 10 mM and about 50 mM (e.g., between about 15 mM and about 45 mM,between about 20 mM and about 40 mM, or between about 25 mM and about 35mM) methionine (or alternatively cysteine or glutathione), between about10 mM and about 50 mM (e.g., between about 15 mM and about 45 mM,between about 20 mM and about 40 mM, or between about 25 mM and about 35mM) histidine, and between about 10 mM and about 50 mM (e.g., betweenabout 15 mM and about 45 mM, between about 20 mM and about 40 mM, orbetween about 25 mM and about 35 mM) sodium ascorbate; or (vii) betweenabout 5 mM to about 45 mM (e.g., between about 10 mM and about 40 mM,between about 15 mM and about 35 mM, or between about 20 mM and about 30mM) sodium ascorbate, between about 5 mM and about 45 mM (e.g., betweenabout 10 mM and about 40 mM, between about 15 mM and about 35 mM, orbetween about 20 mM and about 30 mM) methionine (or alternativelycysteine or glutathione), between about 5 mM and about 45 mM (e.g.,between about 10 mM and about 40 mM, between about 15 mM and about 35mM, or between about 20 mM and about 30 mM) mannitol, and between about5 mM and about 45 mM (e.g., between about 10 mM and about 40 mM, betweenabout 15 mM and about 35 mM, or between about 20 mM and about 30 mM)histidine. In some examples, the liquid is a buffered solution (e.g., aphosphate buffered solution, e.g., a sodium phosphate buffered solution,such as 50 mM sodium phosphate, pH 6.0).

In some examples of any of the compositions provided herein, thecomposition is a solid mixture.

Some embodiments of any of the compositions provided herein can includeat least one antioxidant agent selected from the group of: reducedglutathione, reduced thioredoxin, reduced cysteine, a carotenoid,melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate,bilirubin, uric acid, lipoic acid, a flavonoid, a phenolpropanoid acid,lidocaine, naringenin, fullerene, glucose, mannitol,4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, and dimethylmethoxychromanol. Some embodiments of any of the compositions provided hereincan include at least one (one, two, three, or four) antioxidant agentsselected from mannitol, sodium ascorbate, methionine (or alternativelycysteine or glutathione), and histidine. Some examples of any of thecompositions provided herein include mannitol, sodium ascorbate,methionine (or alternatively cysteine or glutathione), and histidine.Some examples of any of the compositions provided herein can include atleast one chelator selected from the group of:ethylenediaminetetraacetic acid (EDTA), 2,3-dimercapto-1-propanesulfonicacid sodium (DMPS), dimercaptosuccinic acid (DMSA), metallothionin, anddesferroxamine. In some examples of any of the compositions providedherein, the chromatography resin can include at least one resin selectedfrom the group of: anionic exchange chromatography resin, cationicexchange chromatography resin, affinity chromatography resin,hydrophobic interaction chromatography resin, and size exclusionchromatography resin. In some examples of any of the compositionsprovided herein, the resin includes affinity chromatography resinincluding a protein ligand (e.g., protein A or protein G).

Also provided are methods of performing reduced bioburden columnchromatography that includes: (a) providing any of the reduced bioburdenpacked chromatography columns produced by any of the methods describedherein; and (b) performing column chromatography using the reducedbioburden packed chromatography column and reduced bioburden buffer in aclosed system. In some examples of any of the methods described herein,reduced bioburden column chromatography using the reduced bioburdenpacked chromatography column is performed continuously for a period ofat least 4 days (e.g., at least 5 days, at least 7 days, at least 14days, at least 28 days, or at least 60 days). In some embodiments of anyof the methods described herein, the resin in the reduced bioburdenpacked chromatography column in (a) has a percentage binding capacity ofbetween about 65% and about 100% (e.g., between about 75% and about100%) as compared to the same resin not treated with gamma-irradiation.In some embodiments of any of the methods described herein, the resin inthe reduced bioburden packed chromatography column includes at least oneresin selected from the group of: anionic exchange chromatography resin,cationic exchange chromatography resin, affinity or pseudo-affinitychromatography resin, hydrophobic interaction chromatography resin, andsize exclusion chromatography resin. In some examples of any of themethods described herein, the resin includes affinity chromatographyresin including a protein ligand (e.g., protein A or protein G). In someembodiments of any of the methods described herein, the resin includesanionic exchange chromatography resin.

Also provided are integrated, closed or substantially closed, andcontinuous processes for reduced bioburden manufacturing of a purifiedrecombinant protein that include: (a) providing a liquid culture mediumincluding a recombinant protein that is substantially free of cells; and(b) continuously feeding the liquid culture medium into a multi-columnchromatography system (MCCS) including at least one of any of thereduced bioburden packed chromatography columns produced any of themethods described herein; where the process utilizes reduced bioburdenbuffer, is integrated, and runs continuously from the liquid culturemedium to an eluate from the MCCS that is the purified recombinantprotein. In some embodiments of any of the processes described herein,the MCCS performs at least two different unit operations. In someembodiments of any of the processes described herein, the processincludes column switching. In some examples of any of the processesdescribed herein, the MCCS performs the unit operations of capturing therecombinant protein and inactivating viruses, or performs the unitoperations of capturing and purifying the recombinant protein.

In some embodiments of any of the processes described herein, the MCCSincludes at least two reduced bioburden packed chromatography columns.In some examples of any of the processes described herein, the MCCS is aperiodic counter current chromatography system. In some examples of anyof the processes described herein, the MCCS includes a plurality ofcolumns for affinity chromatography, cation exchange chromatography,anion exchange chromatography, or size exclusion chromatography, or anycombination thereof. In some embodiments of any of the methods describedherein, the MCCS includes a column for affinity chromatography, andaffinity chromatography is performed in the process with a capturemechanism selected from the group of: protein A-binding capturemechanism, substrate-binding capture mechanism, antibody- or antibodyfragment-binding capture mechanism, aptamer-binding capture mechanism,and cofactor-binding capture mechanism. In some examples of any of theprocesses described herein, the affinity chromatography is performed inthe process with a protein A-binding capture mechanism, and therecombinant protein is an antibody or an antibody fragment.

Also provided are integrated, closed or substantially closed, andcontinuous processes for reduced bioburden manufacturing of a purifiedrecombinant protein that include: (a) providing a liquid culture mediumincluding a recombinant protein that is substantially free of cells; (b)continuously feeding the liquid culture medium into a first multi-columnchromatography system (MCCS1); (c) capturing the recombinant protein inthe liquid culture medium using the MCCS1; (d) producing an eluate fromthe MCCS1 that includes the recombinant protein and continuously feedingthe eluate into a second multi-column chromatography system (MCCS2); (e)continuously feeding the recombinant protein from the eluate into theMCCS2 and subsequently eluting the recombinant protein to therebyproduce the purified recombinant protein, where: the process utilizesreduced bioburden buffer, is integrated, and runs continuously from theliquid culture medium to the purified recombinant protein, and at leastone column in the MCCS1 and/or MCCS2 contains at least one of any of thereduced bioburden packed chromatography columns produced by any of themethods described herein. In some embodiments of any of the processesdescribed herein, the MCCS1 and/or the MCCS2 performs at least twodifferent unit operations. Some examples of any of the processesdescribed herein involve column switching. In some examples of any ofthe processes described herein, the MCCS1 performs the unit operationsof capturing the recombinant therapeutic protein and inactivatingviruses. In some embodiments of any of the processes provided herein,the MCCS2 performs the unit operations of purifying and polishing therecombinant protein. In some embodiments of any of the processesprovided herein, the MCCS1 and/or MCCS2 includes at least twochromatography columns. In some examples of any of the processesdescribed herein, the MCCS1 is a first periodic counter currentchromatography system (PCCS1). In some examples of any of the processesdescribed herein, the capturing is performed using affinitychromatography, cation exchange chromatography, anion exchangechromatography, or size exclusion chromatography, or any combinationthereof. In some examples of any of the processes described herein, theaffinity chromatography is performed with a capture mechanism selectedfrom the group of: protein A-binding capture mechanism,substrate-binding capture mechanism, antibody- or antibodyfragment-binding capture mechanism, aptamer-binding capture mechanism,and cofactor-binding capture mechanism. In some embodiments of any ofthe processes described herein, the affinity chromatography is performedwith a protein-A binding capture mechanism, and the recombinant proteinis an antibody or an antibody fragment.

In some embodiments of any of the processes described herein, the MCCS2is a second periodic counter current (PCCS2) chromatography system. Inany of the processes described herein, the recombinant protein is atherapeutic recombinant protein. Some embodiments of any of theprocesses described herein further include formulating the purifiedtherapeutic recombinant protein into a pharmaceutical composition. Someembodiments of any of the processes described herein can be performedcontinuously for a period of at least 4 days (e.g., at least 5 days, atleast 7 days, at least 14 days, or at least 28 days).

Also provided are methods of reducing bioburden of a chromatographycomprising (a) exposing a container comprising a substantially drychromatography resin to a dose of gamma-irradiation sufficient to reducethe bioburden of the container and the chromatography resin. Someembodiments of these methods further comprise prior to step (a) drying achromatography resin to remove liquid from the chromatography resin. Insome embodiments, the substantially dry chromatography resin does notcontain a significant amount of an antioxidant agent or a significantamount of a chelator. In some embodiments, the container is a storagevessel. In some embodiments, the chromatography resin is covalentlyattached to a surface of an article (e.g., a chip, membrane, orcassette). In some embodiments, the chromatography resin comprises aprotein ligand (e.g., protein A or protein G). In some embodiments, thechromatography resin is an anionic exchange chromatography resin (e.g.,a chromatography resin comprising N-benzyl-N-methyl-ethanolaminegroups). In some embodiments, the dose is between about 15 kGy to about45 kGy (e.g., between about 20 kGy to about 30 kGy). Also provided arereduced bioburden chromatography resins produced by any of the methodsdescribed herein. In some embodiments, the reduced bioburdenchromatography resin produced has a sterility assurance level (SAL) ofbetween about 1×10⁻⁸ to about 1×10⁻⁵ (e.g., a SAL of between about1×10⁻⁷ to about 1×10⁻⁶). In some embodiments, the reduced chromatographyresin comprises at least one resin selected from the group of: anionicexchange chromatography resin, cationic exchange chromatography resin,affinity chromatography resin, hydrophobic interaction chromatographyresin, and size exclusion chromatography resin. In some embodiments, thereduced chromatography resin comprises an affinity chromatography resincomprising a protein ligand (e.g., protein A). In some embodiments, thereduced chromatography resin comprises an anionic exchangechromatography resin (e.g., an anionic exchange chromatography resincomprising N-benzyl-N-methyl-ethanolamine groups). Also provided aremethods of making a reduced bioburden packed chromatography column thatcomprise providing the reduced bioburden chromatography resin producedby any of the methods described herein; and packing the chromatographyresin into a reduced bioburden column in an aseptic environment. Alsoprovided are reduced bioburden packed chromatography columns produced byany of the methods described herein.

Also provided are integrated, closed, and continuous processes forreduced bioburden manufacturing of a purified recombinant proteincomprising: (a) providing a liquid culture medium comprising arecombinant protein that is substantially free of cells; and (b)continuously feeding the liquid culture medium into a multi-columnchromatography system (MCCS) comprising at least one reduced bioburdenpacked chromatography column produced by any of the methods providedherein; where the process utilizes reduced bioburden buffer, isintegrated, and runs continuously from the liquid culture medium to aneluate from the MCCS that is the purified recombinant protein. Alsoprovided are integrated, closed, and continuous processes for reducedbioburden manufacturing of a purified recombinant protein comprising:(a) providing a liquid culture medium comprising a recombinant proteinthat is substantially free of cells; (b) continuously feeding the liquidculture medium into a first multi-column chromatography system (MCCS1);(c) capturing the recombinant protein in the liquid culture medium usingthe MCCS1; (d) producing an eluate from the MCCS1 that comprises therecombinant protein and continuously feeding the eluate into a secondmulti-column chromatography system (MCCS2); (e) continuously feeding therecombinant protein from the eluate into the MCCS2 and subsequentlyeluting the recombinant protein to thereby produce the purifiedrecombinant protein, wherein: the process utilizes reduced bioburdenbuffer, is integrated, and runs continuously from the liquid culturemedium to the purified recombinant protein, and at least one column inthe MCCS1 and/or MCCS2 contains a reduced bioburden packedchromatography column produced by any of the methods provided herein.

As used herein, the word “a” before a noun represents one or more of theparticular noun. For example, the phrase “a reduced bioburdenchromatography column” represents “one or more reduced bioburdenchromatography columns.”

The term “bioburden” is art known and refers to the level ofself-replicating biological contaminants present in a composition (e.g.,solid or liquid) and/or on the surface (e.g., exterior and/or interiorsurface) of an article(s). For example, bioburden can refer toself-replicating biological contaminants present in a compositioncontaining a chromatography resin or a packed chromatography resin(e.g., self-replicating biological contaminants present in a packedchromatography resin in a packed chromatography column). In otherexamples, bioburden can to refer to self-replicating biologicalcontaminants on the inner surface of a chromatography column and/orwithin the chromatography resin within the chromatography column (e.g.,biological contaminants on the inner surface of a chromatography columnand biological contaminants in the packed chromatography resin withinthe chromatography column). Bioburden can also refer to theself-replicating biological contaminants present within a liquid (e.g.,a buffer used in any of the methods or processes described herein).Non-limiting examples of self-replicating biological contaminants can bebacteria (e.g., Gram-positive or Gram-negative bacteria, or a bacterialspore), mycobacteria, viruses (e.g., a vesivirus, a Cache Valley virus,a parvovirus, a herpes virus, and a bunyavirus), parasites, fungi,yeast, and protozoa. Exemplary methods for determining bioburden aredescribed herein. Additional methods for determining bioburden are knownin the art.

The term “reducing bioburden” is art known and refers to a decrease(e.g., a detectable decrease) in the level of self-replicatingbiological contaminants present in a composition (e.g., solid or liquid)and/or on the surface (e.g., exterior and/or interior surface) of anarticle(s). Non-limiting examples of methods for reducing bioburden of achromatography resin (e.g., packed chromatography resin), buffer, and/ora chromatography column (e.g., a packed chromatography column) aredescribed herein. Additional methods for reducing bioburden of any ofthe compositions described herein are known in the art.

The term “reduced bioburden chromatography resin” means a chromatographyresin that has been treated to decrease the level of self-replicatingbiological contaminants present in the chromatography resin (e.g., adetectable decrease in the level of self-replicating biologicalcontaminants present in a composition containing a chromatography resin,e.g., a slurry). For example, reduced bioburden chromatography resin canbe a resin exposed to gamma-irradiation in a dose sufficient to decreasethe level of self-replicating biological contaminants in thechromatography resin (e.g., a composition containing a chromatographyresin exposed to gamma-irradiation in a dose sufficient to decrease thelevel of self-replicating biological contaminants in the chromatographyresin). For example, a reduced bioburden chromatography resin can be aresin that has been exposed to a dose of between about 1 kGy to about 15kGy, between about 1 kGy and about 20 kGy gamma-irradiation, betweenabout 1 kGy and about 25 kGy gamma-irradiation, between about 1 kGy andabout 30 kGy gamma-irradiation, or between about 1 kGy and about 35 kGygamma-irradiation. Exemplary methods for reducing bioburden of achromatography resin are described herein. Additional methods forreducing the bioburden of a chromatography resin are known in the art.

The term “reduced bioburden chromatography column” means achromatography column (e.g., a packed chromatography column) thatincludes a treated chromatography resin (e.g., gamma-irradiatedchromatography resin), that has a level of self-replicating biologicalcontaminants that is less than the level of self-replicating biologicalcontaminants present in an identical chromatography column that includesan untreated chromatography resin. For example, a reduced bioburdenchromatography column can include a treated chromatography resin havinga sterility assurance level of at least or about 1×10⁻⁶, 1×10⁻⁷, 1×10⁻⁸,1×10⁻⁹, or 1×10⁻¹⁰.

The term “reduced bioburden buffer” is art known and means a treated(e.g., filtered, autoclaved, and/or gamma-irradiated) liquid (e.g., atreated buffered solution) that has a level of self-replicatingcontaminating agent(s) that is less than the level of self-replicatingcontaminating agent(s) found in an identical untreated liquid. Forexample, a reduced bioburden buffer can have a sterility assurance levelof at least or about 1×10⁻⁶, 1×10⁻⁷, 1×10⁻⁸, 1×10⁻⁹, or 1×10⁻¹⁰.

“Absolute sterility” or “absolutely sterile” are terms used to describea composition or process that is/are completely free of self-replicatingbiological contaminants. For example, the term can apply to agamma-irradiated chromatography resin, the interior surface and contents(e.g., chromatography resin) of a chromatography column, and/or abuffer. An absolutely sterile composition or process can be clean (asthat term is known in the art).

“Sterile” or “sterility” are terms used to describe a composition orprocess that have a sterility assurance level of about or less than1.0×10⁻⁶ (e.g., about or less than 1.0×10⁻⁷, about or less than1.0×10⁻⁸, about or less than 1.0×10⁻⁹, or 1×10⁻¹⁰). The determination ofwhether a composition or process is sterile can be tested using a numberof validated production processes known in the art. For example, asterile composition or process can be completely free of viableself-replicating biological contaminants (e.g., any of theself-replicating biological contaminants described herein). A sterilecomposition or process can also be clean (as that term is known in theart).

The term “sterilization” means a validated process used to render acomposition sterile (as defined herein). The inactivation rate ofresistant indicator self-replicating biological contaminants (e.g.,bacteria) during a treatment process can be measured in order todetermine whether sterility (as defined herein) has been achieved for acomposition.

The term “sterility assurance level” or “SAL” is art-known and means alevel of confidence of achieving absolute sterility within a batch oftreated units. The probability is usually calculated based on theresults of inactivation studies performed during validation andexpressed in the form of 1×10^(−n).

The term “aseptic” is used to describe a composition or process that isfree of disease-causing or symptom-causing self-replicating biologicalcontaminants (e.g., any of the self-replicating biological contaminantsdescribed herein). An aseptic composition or process can also be clean(as that term is known in the art).

The term “unit operation” is a term of art and means a functional stepthat can be performed in a process of purifying a recombinant proteinfrom a liquid culture medium. For example, a unit of operation can befiltering (e.g., removal of contaminant bacteria, yeast, viruses, and/ormycobacteria, and/or particulate matter from a fluid including arecombinant protein), capturing, epitope tag removal, purifying, holdingor storing, polishing, virus inactivating, adjusting the ionicconcentration and/or pH of a fluid including the recombinant protein,and removing unwanted salts.

The term “capturing” means a step performed to partially purify orisolate (e.g., at least or about 5%, e.g., at least or about 10%, 15%,20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, orat least or about 95% pure by weight) and concentrate a recombinantprotein (e.g., a recombinant therapeutic protein) from one or more othercomponents present in a liquid culture medium or a diluted liquidculture medium (e.g., culture medium proteins or one or more othercomponents (e.g., DNA, RNA, or other proteins) present in or secretedfrom a mammalian cell). Typically, capturing is performed using achromatography resin that binds a recombinant protein (e.g., through theuse of affinity chromatography). Non-limiting methods for capturing arecombinant protein from a liquid culture medium or diluted liquidculture medium are described herein and others are known in the art. Arecombinant protein can be captured from a liquid culture medium usingat least one chromatography column and/or chromatographic membrane(e.g., any of the chromatography columns and/or chromatographicmembranes described herein).

The term “purifying” means a step performed to isolate a recombinantprotein (e.g., a recombinant therapeutic protein) from one or more otherimpurities (e.g., bulk impurities) or components present in a fluidincluding a recombinant protein (e.g., liquid culture medium proteins orone or more other components (e.g., DNA, RNA, other proteins,endotoxins, viruses, etc.) present in or secreted from a mammaliancell). For example, purifying can be performed during or after aninitial capturing step. Purification can be performed using achromatography resin, membrane, or any other solid support that bindseither a recombinant protein or contaminants (e.g., through the use ofaffinity chromatography, hydrophobic interaction chromatography, anionor cation exchange chromatography, or molecular sieve chromatography). Arecombinant protein can be purified from a fluid including therecombinant protein using at least one chromatography column and/orchromatographic membrane (e.g., any of the chromatography columns orchromatographic membranes described herein).

The term “polishing” is a term of art and means a step performed toremove remaining trace or small amounts of contaminants or impuritiesfrom a fluid including a recombinant protein (e.g., a recombinanttherapeutic protein) that is close to a final desired purity. Forexample, polishing can be performed by passing a fluid including therecombinant protein through a chromatographic column(s) or membraneabsorber(s) that selectively binds to either the target recombinantprotein or small amounts of contaminants or impurities present in afluid including a recombinant protein. In such an example, theeluate/filtrate of the chromatographic column(s) or membrane absorber(s)includes the recombinant protein.

The term “filtering” means the removal of at least part of (e.g., atleast 80%, 90%, 95%, 96%, 97%, 98%, or 99%) undesired biologicalcontaminants (e.g., a mammalian cell, bacteria, yeast cells, viruses, ormycobacteria) and/or particulate matter (e.g., precipitated proteins)from a liquid (e.g., a liquid culture medium or fluid present in any ofthe processes described herein).

The term “eluate/filtrate” is a term of art and means a fluid that isemitted from a chromatography column or chromatographic membrane thatincludes a detectable amount of a recombinant protein (e.g., arecombinant therapeutic protein).

The term “integrated process” means a process which is performed usingstructural elements that function cooperatively to achieve a specificresult (e.g., the purification of a recombinant protein from a liquidculture medium).

The term “continuous process” means a process which continuously feedsfluid through at least a part of the system. For example, a continuousprocess is a process which continuously feeds a liquid culture mediumincluding a recombinant protein from a bioreactor through a MCCS.Another example of a continuous process is a process which continuouslyfeeds a liquid culture medium including a recombinant protein from abioreactor through a first and second MCCS (MCCS1 and MCCS2). Additionalexamples include a process which continuously feeds a liquid culturemedium including a recombinant protein through a MCCS, a process thatcontinuously feeds a liquid culture medium including a recombinantprotein through a MCCS1 and a MCCS2, or a process that continuouslyfeeds a fluid including a recombinant protein through a MCCS2.

The term “closed process” is a term of art and means a process that isperformed such that components of the process (e.g., chromatographyresins and/or buffers) that come into contact with the recombinantprotein or liquids including the recombinant protein are notintentionally exposed to contaminating agents for a significant periodof time (e.g., not intentionally air-exposed for a significant period oftime).

The term “therapeutic protein drug substance” means a recombinantprotein (e.g., an immunoglobulin, protein fragment, engineered protein,or enzyme) that has been sufficiently purified or isolated fromcontaminating proteins, lipids, and nucleic acids (e.g., contaminatingproteins, lipids, and nucleic acids present in a liquid culture mediumor from a host cell (e.g., from a mammalian, yeast, or bacterial hostcell)) and biological contaminants (e.g., viral and bacterialcontaminants), and can be formulated into a pharmaceutical agent withoutany further substantial purification and/or decontamination step(s).

The term “multi-column chromatography system” or “MCCS” means a systemof a total of two or more interconnected or switching chromatographycolumns and/or chromatographic membranes. A non-limiting example of amulti-column chromatography system is a periodic counter currentchromatography system (PCC) including a total of two or moreinterconnected or switching chromatography columns and/orchromatographic membranes. Additional examples of multi-columnchromatography systems are described herein and are known in the art.

The term “substantially free” means a composition (e.g., a liquidculture medium) that is at least or about 90% free (e.g., at least orabout 95%, 96%, 97%, 98%, or at least or about 99% free, or about 100%free) of a specified substance (e.g., a mammalian cell or acontaminating protein, nucleic acid, carbohydrate, or lipid form amammalian cell).

The term “mammalian cell” means any cell from or derived from any mammal(e.g., a human, a hamster, a mouse, a green monkey, a rat, a pig, a cow,or a rabbit). For example, a mammalian cell can be an immortalized cell.In some embodiments, the mammalian cell is a differentiated cell. Insome embodiments, the mammalian cell is an undifferentiated cell.Non-limiting examples of mammalian cells are described herein.Additional examples of mammalian cells are known in the art.

The term “culturing” or “cell culturing” means the maintenance orproliferation of a mammalian cell under a controlled set of physicalconditions.

The term “culture of mammalian cells” means a liquid culture mediumincluding a plurality of mammalian cells that is maintained orproliferated under a controlled set of physical conditions.

The term “liquid culture medium” means a fluid that includes sufficientnutrients to allow a cell (e.g., a mammalian cell) to grow orproliferate in vitro. For example, a liquid culture medium can includeone or more of: amino acids (e.g., 20 amino acids), a purine (e.g.,hypoxanthine), a pyrimidine (e.g., thymidine), choline, inositol,thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine,riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium,glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate.In some embodiments, a liquid culture medium can include serum from amammal. In some embodiments, a liquid culture medium does not includeserum or another extract from a mammal (a defined liquid culturemedium). In some embodiments, a liquid culture medium can include tracemetals, a mammalian growth hormone, and/or a mammalian growth factor.Another example of liquid culture medium is minimal medium (e.g., amedium including only inorganic salts, a carbon source, and water).Non-limiting examples of liquid culture medium are described herein.Additional examples of liquid culture medium are known in the art andare commercially available. A liquid culture medium can include anydensity of mammalian cells. For example, as used herein, a volume ofliquid culture medium removed from a bioreactor can be substantiallyfree of mammalian cells.

The term “animal-derived component free liquid culture medium” means aliquid culture medium that does not include any components (e.g.,proteins or serum) derived from a mammal.

The term “serum-free liquid culture medium” means a liquid culturemedium that does not include a mammalian serum.

The term “serum-containing liquid culture medium” means a liquid culturemedium that includes a mammalian serum.

The term “chemically-defined liquid culture medium” is a term of art andmeans a liquid culture medium in which all of the chemical componentsare known. For example, a chemically-defined liquid culture medium doesnot include fetal bovine serum, bovine serum albumin, or human serumalbumin, as these preparations typically include a complex mix ofalbumins and lipids.

The term “protein-free liquid culture medium” means a liquid culturemedium that does not include any protein (e.g., any detectable protein).

The term “immunoglobulin” means a polypeptide including an amino acidsequence of at least 15 amino acids (e.g., at least 20, 30, 40, 50, 60,70, 80, 90, or 100 amino acids) of an immunoglobulin protein (e.g., avariable domain sequence, a framework sequence, and/or a constant domainsequence). The immunoglobulin may, for example, include at least 15amino acids of a light chain immunoglobulin, e.g., at least 15 aminoacids of a heavy chain immunoglobulin. The immunoglobulin may be anisolated antibody (e.g., an IgG, IgE, IgD, IgA, or IgM), e.g., asubclass of IgG (e.g., IgG1, IgG2, IgG3, or IgG4). The immunoglobulinmay be an antibody fragment, e.g., a Fab fragment, a F(ab′)₂ fragment,or an a scFv fragment. The immunoglobulin may also be a bi-specificantibody or a tri-specific antibody, or a dimer, trimer, or multimerantibody, or a diabody, an Affibody®, or a Nanobody®. The immunoglobulincan also be an engineered protein including at least one immunoglobulindomain (e.g., a fusion protein). Non-limiting examples ofimmunoglobulins are described herein and additional examples ofimmunoglobulins are known in the art.

The term “protein fragment” or “polypeptide fragment” means a portion ofa polypeptide sequence that is at least or about 4 amino acids, at leastor about 5 amino acids, at least or about 6 amino acids, at least orabout 7 amino acids, at least or about 8 amino acids, at least or about9 amino acids, at least or about 10 amino acids, at least or about 11amino acids, at least or about 12 amino acids, at least or about 13amino acids, at least or about 14 amino acids, at least or about 15amino acids, at least or about 16 amino acids, at least or about 17amino acids, at least or about 18 amino acids, at least or about 19amino acids, or at least or about 20 amino acids in length, or more than20 amino acids in length. A recombinant protein fragment can be producedusing any of the processes described herein.

The term “engineered protein” means a polypeptide that is not naturallyencoded by an endogenous nucleic acid present within an organism (e.g.,a mammal). Examples of engineered proteins include enzymes (e.g., withone or more amino acid substitutions, deletions, insertions, oradditions that result in an increase in stability and/or catalyticactivity of the engineered enzyme), fusion proteins, antibodies (e.g.,divalent antibodies, trivalent antibodies, or a diabody), andantigen-binding proteins that include at least one recombinantscaffolding sequence.

The term “secreted protein” or “secreted recombinant protein” means aprotein (e.g., a recombinant protein) that originally included at leastone secretion signal sequence when it is translated within a mammaliancell, and through, at least in part, enzymatic cleavage of the secretionsignal sequence in the mammalian cell, is secreted at least partiallyinto the extracellular space (e.g., a liquid culture medium). Skilledpractitioners will appreciate that a “secreted” protein need notdissociate entirely from the cell to be considered a secreted protein.

The term “perfusion bioreactor” means a bioreactor including a pluralityof cells (e.g., mammalian cells) in a first liquid culture medium,wherein the culturing of the cells present in the bioreactor includesperiodic or continuous removal of the first liquid culture medium and atthe same time or shortly thereafter adding substantially the same volumeof a second liquid culture medium to the bioreactor. In some examples,there is an incremental change (e.g., increase or decrease) in thevolume of the first liquid culture medium removed and added overincremental periods (e.g., an about 24-hour period, a period of betweenabout 1 minute and about 24-hours, or a period of greater than 24 hours)during the culturing period (e.g., the culture medium refeed rate on adaily basis). The fraction of media removed and replaced each day canvary depending on the particular cells being cultured, the initialseeding density, and the cell density at a particular time. “RV” or“reactor volume” means the volume of the culture medium present at thebeginning of the culturing process (e.g., the total volume of theculture medium present after seeding).

The term “fed-batch bioreactor” is a term of art and means a bioreactorincluding a plurality of cells (e.g., mammalian cells) in a first liquidculture medium, wherein the culturing of the cells present in thebioreactor includes the periodic or continuous addition of a secondliquid culture medium to the first liquid culture medium withoutsubstantial or significant removal of the first liquid culture medium orsecond liquid culture medium from the cell culture. The second liquidculture medium can be the same as the first liquid culture medium. Insome examples of fed-batch culture, the second liquid culture medium isa concentrated form of the first liquid culture medium. In some examplesof fed-batch culture, the second liquid culture medium is added as a drypowder.

The term “clarified liquid culture medium” means a liquid culture mediumobtained from a bacterial or yeast cell culture that is substantiallyfree (e.g., at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% free) ofbacteria or yeast cells.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the static binding curve of resin (mg/mLprotein) for untreated GE Mab Select SuRe™ resin, 25 kGygamma-irradiated GE Mab Select SuRe™ resin, 15 kGy gamma-irradiated JSRLifeSciences Amsphere ProA JWT203 resin, and 15 kGy gamma-irradiatedKaneka KanCap A resin.

FIG. 2 is a table showing the 280 nm absorbance (protein A absorbance)of supernatant from GE Mab Select SuRe™ resin following 30 kGygamma-irradiation and from JSR LifeSciences Amsphere ProA JWT203 resinfollowing 30 kGy gamma-irradiation.

FIG. 3 is a graph showing the amount of protein in eluate over multiplecycles of chromatography performed using untreated JSR LifeSciencesAmsphere ProA JWT203 resin (virgin eluate) or 15 kGy gamma-irradiatedJSR LifeSciences Amsphere ProA JWT203 resin (gamma eluate).

FIG. 4 is a chromatograph showing the elution profile of cycle 7 andcycle 11 during continuous chromatography performed using untreated JSRLifeSciences Amsphere ProA JWT203 resin.

FIG. 5 is a chromatograph showing the elution profile of cycle 7 andcycle 11 during continuous chromatography performed using 15 kGygamma-irradiated JSR LifeSciences Amsphere ProA JWT203 resin.

FIG. 6 is a graph showing the amount of protein in the eluate overmultiple cycles of continuous chromatography performed using untreatedduring continuous chromatography performed using untreated JSRLifeSciences Amsphere ProA JWT203 resin (virgin) or 15 kGygamma-irradiated JSR LifeSciences Amsphere ProA JWT203 resin (Gamma).

FIG. 7 is a graph of the protein bound over multiple cycles ofchromatography performed using untreated JSR LifeSciences Amsphere ProAJWT203 resin (virgin) or 15 kGy gamma-irradiated JSR LifeSciencesAmsphere ProA JWT203 resin (gamma).

FIG. 8 is a graph showing the amount of protein in eluate over multiplecycles of chromatography performed using untreated JSR LifeSciencesAmsphere ProA JWT203 resin (virgin) or 29 kGy gamma-irradiated JSRLifeSciences Amsphere ProA JWT203 resin (Gamma).

FIG. 9 is a graph showing the amount of protein (anti-αβTCR (IgG1) oranti-TGFβ (IgG4)) in eluate over multiple cycles of chromatographyperformed using untreated JSR LifeSciences Amsphere ProA JWT203 resin(virgin), 15 kGy gamma-irradiated JSR LifeSciences Amsphere ProA JWT203resin (15 kGy), or 29 kGy gamma-irradiated JSR LifeSciences AmsphereProA JWT203 resin (29 kGy).

FIG. 10 is a list of exemplary recombinant proteins that are enzymesthat can be used to treat specific disorders in humans.

FIG. 11 is a graph of the protein in the eluate of untreated (virgin)and 29-kGy gamma-irradiated GE Mab Select SuRe™ resin over multiplechromatography cycles when a culture medium containing monoclonal IgG1was used to load each resin.

FIG. 12 is a graph of amount of protein bound to untreated (virgin) and29-kGy gamma-irradiated GE Mab Select SuRe™ resin over multiplechromatography cycles when a culture medium containing monoclonal IgG1was used to load each resin.

FIG. 13 is a graph of the amount of protein bound (mg bound protein/mLof resin) to untreated (virgin) and 29-kGy gamma-irradiated GE MabSelect SuRe™ LX resin following loading with different equilibriumconcentrations of a monoclonal IgG4 antibody.

FIG. 14 is a graph of the static binding capacity (mg protein/mL ofresin) at an equilibrium IgG4 antibody concentration of 2 mg/mL ofuntreated GE Mab Select SuRe™ LX resin (virgin LX), 29-kGygamma-irradiated GE Mab Select SuRe™ LX resin (29kGY LX), dry untreatedGE Mab Select SuRe™ LX resin, and dry 29-kGy gamma-irradiated GE MabSelect SuRe™ LX resin, and GE Mab Select SuRe™ LX resin gamma-irradiatedat a dose of 29 kGy in the presence of one of seven different testedbuffers containing at least one antioxidant agent.

FIG. 15 is a graph of the amount of protein in the eluate over multiplechromatography cycles performed using untreated GE Mab Select SuRe™ LXresin (virgin) or GE Mab Select SuRe™ LX resin gamma-irradiated with adose of 29 kGy in the presence of 25 mM mannitol, 25 mM histidine, 25 mMsodium ascorbate, 25 mM methionine, 50 mM sodium phosphate, pH 6.0.

FIG. 16 is a graph of the amount of protein bound over multiplechromatography cycles performed using untreated GE Mab Select SuRe™ LXresin (virgin) or GE Mab Select SuRe™ LX resin gamma-irradiated with adose of 29 kGy in the presence of 25 mM mannitol, 25 mM histidine, 25 mMsodium ascorbate, 25 mM methionine, 50 mM sodium phosphate, pH 6.0.

FIG. 17 is a non-reduced sodium-dodecyl sulfate polyacrylamide gelshowing the IgG1 antibody eluate over multiple chromatography cyclesusing untreated GE Mab Select SuRe™ LX resin (virgin LX) and GE MabSelect SuRe™ LX resin gamma-irradiated with a dose of 29 kGy in thepresence of 25 mM mannitol, 25 mM histidine, 25 mM sodium ascorbate, 25mM methionine, 50 mM sodium phosphate, pH 6.0.

DETAILED DESCRIPTION

Provided herein are methods of reducing bioburden of a chromatographyresin that include exposing a container including a compositionincluding a chromatography resin and at least one antioxidant agentand/or chelator to a dose of gamma-irradiation sufficient to reduce thebioburden of the container and the chromatography resin, where the atleast one antioxidant agent and/or chelator are present in an amountsufficient to ameliorate the loss of binding capacity of thechromatography resin after/upon exposure to the dose ofgamma-irradiation. Also provided are reduced bioburden chromatographycolumns containing a reduced bioburden chromatography resin prepared byany of the methods described herein, compositions including achromatography resin and at least one chelator and/or antioxidant agent,methods of performing reduced bioburden column chromatography using atleast one of these reduced bioburden chromatography columns, andintegrated, closed or substantially closed, and continuous processes forreduced bioburden manufacturing of a purified recombinant protein thatinclude the use of at least one of these reduced bioburdenchromatography columns. Non-limiting aspects of these methods andprocesses are described below. As can be appreciated in the art, thevarious aspects described below can be used in any combination withoutlimitation.

Compositions Containing Chromatography Resin and an Antioxidant and/orChelator

Provided herein are compositions including a chromatography resin (e.g.,any of the chromatography resins described herein or known in the art)and at least one antioxidant agent and/or chelator (e.g., any of theantioxidant agents and/or chelators described herein or known in theart), wherein the at least one antioxidant agent and/or chelator ispresent in an amount sufficient to ameliorate the loss of bindingcapacity of the chromatography resin upon treatment with a dose ofgamma-irradiation sufficient to reduce bioburden of the composition. Forexample, the chromatography resin can be at least one of anionicexchange chromatography resin, cationic exchange chromatography resin,affinity or pseudo-affinity chromatography resin, hydrophobicinteraction chromatography resin, and size exclusion chromatographyresin, or any combination thereof. In some examples, the chromatographyresin is a resin including a protein or peptide ligand (e.g., anaffinity chromatography resin with a protein or peptide ligand, e.g., aprotein A or protein G chromatography resin).

The composition, e.g., can be a slurry of sedimented chromatographyresin in a liquid including the at least one antioxidant agent and/orchelator. For example, the liquid can contain at least one (e.g., one,two, three, or four) of methionine (or alternatively cysteine orglutathione), sodium ascorbate, histidine, and mannitol. In someexamples, the liquid contains methionine (or alternatively cysteine orglutathione), sodium ascorbate, histidine, and mannitol. In someexamples, the liquid comprises: (i) between 75 mM and about 125 mM(e.g., between 80 mM and about 120 mM, between about 85 mM and about 115mM, between about 90 mM and about 110 mM, or between about 95 mM andabout 105 mM) mannitol; (ii) between 75 mM and about 125 mM (e.g.,between about 80 mM and about 120 mM, between about 85 mM and about 115mM, between about 90 mM and about 110 mM, or between about 95 mM andabout 105 mM) methionine (or alternatively cysteine or glutathione);(iii) between 75 mM and about 125 mM (e.g., between about 80 mM andabout 120 mM, between about 85 mM and about 115 mM, between about 90 mMand about 110 mM, or between about 95 mM and about 105 mM) sodiumascorbate; (iv) between 75 mM and about 125 mM (e.g., between about 80mM and about 120 mM, between about 85 mM and about 115 mM, between about90 mM and about 110 mM, or between about 95 mM and about 105 mM)histidine; (v) between about 30 mM and about 70 mM (e.g., between about35 mM and about 65 mM, between about 40 mM and about 60 mM, or betweenabout 45 mM and about 55 mM) methionine (or alternatively cysteine orglutathione) and between about 30 mM and about 70 mM (e.g., betweenabout 35 mM and about 65 mM, between about 40 mM and about 60 mM, orbetween about 45 mM and about 55 mM) histidine; (vi) between about 10 mMand about 50 mM (e.g., between about 15 mM and about 45 mM, betweenabout 20 mM and about 40 mM, or between about 25 mM and about 35 mM)methionine (or alternatively cysteine or glutathione), between about 10mM and about 50 mM (e.g., between about 15 mM and about 45 mM, betweenabout 20 mM and about 40 mM, or between about 25 mM and about 35 mM)histidine, and between about 10 mM and about 50 mM (e.g., between about15 mM and about 45 mM, between about 20 mM and about 40 mM, or betweenabout 25 mM and about 35 mM) sodium ascorbate; or (vii) between about 5mM to about 45 mM (e.g., between about 10 mM and about 40 mM, betweenabout 15 mM and about 35 mM, or between about 20 mM and about 30 mM)sodium ascorbate, between about 5 mM and about 45 mM (e.g., betweenabout 10 mM and about 40 mM, between about 15 mM and about 35 mM, orbetween about 20 mM and about 30 mM) methionine (or alternativelycysteine or glutathione), between about 5 mM and about 45 mM (e.g.,between about 10 mM and about 40 mM, between about 15 mM and about 35mM, or between about 20 mM and about 30 mM) mannitol, and between about5 mM and about 45 mM (e.g., between about 10 mM and about 40 mM, betweenabout 15 mM and about 35 mM, or between about 20 mM and about 30 mM)histidine. In some examples, the liquid is a buffered solution (e.g., aphosphate buffered solution, e.g., a sodium phosphate buffered solution,such as 50 mM sodium phosphate, pH 6.0). In some examples, the liquid isa buffered solution (e.g., a phosphate buffered solution, such as asodium phosphate buffered solution (e.g., 50 mM sodium phosphate, pH6.0).

In some examples, the composition can be a solid mixture (e.g., a drysolid mixture or a wetted or moist solid mixture). The solid mixture caninclude at least one (e.g., one, two, three, or four) of methionine (oralternatively cysteine or glutathione), sodium ascorbate, histidine, andmannitol. In some examples, the solid mixture contains methionine (oralternatively cysteine or glutathione), sodium ascorbate, histidine, andmannitol. In some examples, the composition is a chromatography resinpacked in liquid including the at least one antioxidant agent and/orchelator. Non-limiting examples of such liquids are described herein.

Also provided herein is a container (e.g., a plastic container or achromatography column) including a chromatography resin (e.g., any ofthe chromatography resins described herein or known in the art) and atleast one antioxidant agent and/or chelator (e.g., any of theantioxidant agents and/or chelators described herein or known in theart), wherein the at least one antioxidant agent and/or chelator ispresent in an amount sufficient to ameliorate the loss of bindingcapacity of the chromatography resin upon treatment with a dose ofgamma-irradiation sufficient to reduce bioburden of the composition. Forexample, the container (e.g., a plastic container or a chromatographycolumn) can have an internal volume of, e.g., at least about 1 mL, 5 mL,at least about 10 mL, at least about 20 mL, at least about 30 mL, atleast about 40 mL, at least about 50 mL, at least about 60 mL, at leastabout 70 mL, at least about 80 mL, at least about 90 mL, at least about100 mL, at least about 110 mL, at least about 120 mL, at least about 130mL, at least about 140 mL, at least about 150 mL, at least about 160 mL,at least about 170 mL, at least about 180 mL, at least about 190 mL, atleast about 200 mL, at least about 210 mL, at least about 220 mL, atleast about 230 mL, at least about 240 mL, at least about 250 mL, atleast 300 mL, at least 350 mL, at least 400 mL, or at least 500 mL. Forexample, a container can have an internal volume of, e.g., between about1 mL and about 500 mL, between about 1 mL and about 50 mL, between about5 mL and about 500 mL, between about 5 mL and about 400 mL, betweenabout 5 mL and about 350 mL, between about 5 mL and about 300 mL,between about 5 mL and about 250 mL, between about 5 mL and about 200mL, between about 5 mL and about 150 mL, between about 5 mL and about100 mL, or between about 5 mL and about 50 mL. In some examples, thechromatography resin in the container is a slurry of sedimentedchromatography resin in a liquid including the at least one antioxidantagent and/or chelator. In some examples, the container includes a packedchromatography resin (e.g., packed in a liquid including the at leastone antioxidant agent and/or chelator).

Any of the compositions provided herein can contain at least one (e.g.,2, 3, 4, 5, 6, 7, 8, 9, or 10) antioxidant agent and/or at least one(e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) chelator. Any of the antioxidantagent(s) included in any of the compositions provided herein can havethe ability to quench one or more of the following reactive oxygenand/or nitrogen species: hydroxyl radical, carbonate radical, superoxideanion, peroxyl radical, peroxynitrite, nitrogen dioxide, and nitricoxide. Non-limiting examples of antioxidant agents that can be includedin any of the compositions provided herein include: reduced glutathione,reduced thioredoxin, reduced cysteine, a carotenoid, melatonin,lycopene, tocopherol, reduced ubiquinone, ascorbate, bilirubin, uricacid, lipoic acid, a flavonoid, a phenolpropanoid acid, lidocaine,naringenin, fullerene, glucose, mannitol,4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, and dimethylmethoxychromanol. Additional non-limiting examples of antioxidant agentsinclude antioxidant enzymes (e.g., superoxide dismutase, glutathioneperoxidase, glutathione reductase, catalase, and thioredoxin reductase).Additional examples of antioxidant agents that can be included in any ofthe compositions provided herein include mannitol, sodium ascorbate,methionine, and histidine. Further examples of antioxidant agentsinclude cysteine, taurine, mercaptopropionylglycine, N-acetylcysteine,garlic oil, diallylsulfide, dihydrolipoic acid, and diallyltrisulfide.Some embodiments that include an antioxidant enzyme as an antioxidantagent can further include one or more substrate(s) for the enzyme. Anantioxidant agent can be identified using a number of methods known inthe art including, for example, spin trapping, redox sensitive dyes, andchemiluminescence assays.

Any of the chelator(s) included in any of the compositions providedherein can have the ability to bind a redox active metal (e.g., Cu²⁺ andFe²⁺) with high affinity (e.g., about or less than 1 μM, about or lessthan 800 nM, about or less than 700 nM, about or less than 600 nM, aboutor less than 500 nM, about or less than 400 nM, about or less than 300nM, about or less than 250 nM, about or less than 200 nM, about or lessthan 150 nM, about or less than 100 nM, about or less than 80 nM, aboutor less than 60 nM, about or less than 40 nM, about or less than 20 nM,or about or less than 1 nm). Non-limiting examples of chelator(s) thatcan be included in any of the compositions provided herein includeethylenediaminetetraacetic acid (EDTA), 2,3-dimercapto-1-propanesulfonicacid sodium (DMPS), dimercaptosuccinic acid (DMSA), metallothionin, anddesferroxamine.

The concentration of each of the chelator(s) and/or antioxidant(s) inany of the compositions provided herein can be between about 0.1 mM andabout 150 mM (e.g., between about 0.1 mM and about 150 mM, between about0.1 mM and about 125 mM, between about 0.1 mM and about 100 mM, betweenabout 0.1 mM and about 80 mM, between about 0.1 mM and about 60 mM,between about 0.1 mM and about 50 mM, between about 0.1 mM and about 40mM, between about 0.1 mM and about 30 mM, between about 0.1 mM and about25 mM, between about 0.1 mM and about 20 mM, between about 0.1 mM andabout 10 mM, between about 0.1 mM and about 5.0 mM, between about 0.5 mMand about 150 mM, between about 0.5 mM and about 100 mM, between about0.5 mM and about 50 mM, between about 0.5 mM and about 25 mM, betweenabout 0.5 mM and about 15 mM, between about 0.5 mM and about 10 mM,between about 0.5 mM and about 5 mM, between about 1 mM and about 125mM, between about 1 mM and about 120 mM, between about 1 mM and about100 mM, between about 1 mM and about 80 mM, between about 1 mM and about60 mM, between about 1 mM and about 50 mM, between about 1 mM and about40 mM, between about 1 mM and about 30 mM, between about 1 mM and about25 mM, between about 5 mM and about 150 mM, between about 5 mM and about125 mM, between about 5 mM and about 100 mM, between about 5 mM andabout 80 mM, between about 5 mM and about 60 mM, between about 5 mM andabout 50 mM, between about 5 mM and about 40 mM, between about 5 mM andabout 30 mM, between about 5 mM and about 25 mM, between about 10 mM andabout 150 mM, between about 10 mM and about 125 mM, between about 1 mMand about 100 mM, between about 10 mM and about 80 mM, between about 10mM and about 60 mM, between about 10 mM and about 50 mM, between about10 mM and about 40 mM, between about 10 mM and about 30 mM, betweenabout 10 mM and about 25 mM, between about 20 mM and about 150 mM,between about 20 mM and about 125 mM, between about 20 mM and about 100mM, between about 20 mM and about 80 mM, between about 20 mM and about60 mM, between about 20 mM and about 50 mM, between about 20 mM andabout 40 mM, between about 20 mM and about 30 mM, between about 30 mMand about 150 mM, between about 30 mM and about 125 mM, between about 30mM and about 100 mM, between about 30 mM and about 80 mM, between about30 mM and about 60 mM, between about 30 mM and about 50 mM, betweenabout 30 mM and about 40 mM, between about 40 mM and about 150 mM,between about 40 mM and about 125 mM, between about 40 mM and about 100mM, between about 40 mM and about 90 mM, between about 40 mM and about80 mM, between about 40 mM and about 70 mM, between about 40 mM andabout 60 mM, between about 50 mM and about 150 mM, between about 50 mMand about 125 mM, between about 50 mM and about 100 mM, between about 50mM and about 80 mM, between about 50 mM and about 60 mM, between about80 mM and about 150 mM, between about 80 mM and about 125 mM, betweenabout 80 mM and about 100 mM, between about 100 mM and about 150 mM, orbetween about 100 mM and about 125 mM).

In some examples, the compositions provided herein contain one or moreof 5 mM to about 150 mM mannitol (e.g., between about 10 mM and about150 mM, between about 20 mM and about 150 mM, between about 30 mM andabout 150 mM, between about 40 mM and about 150 mM, between about 50 mMand about 150 mM, between about 60 mM and about 140 mM, between about 70mM and about 130 mM, between about 80 mM and about 120 mM, between about90 mM and about 110 mM, between about 95 mM and about 105 mM, betweenabout 5 mM and about 50 mM, between about 5 mM and about 45 mM, betweenabout 5 mM and about 40 mM, between about 5 mM and about 35 mM, betweenabout 10 mM and about 35 mM, between about 15 mM and about 35 mM, orbetween about 20 mM and about 30 mM mannitol); 5 mM to about 150 mM(e.g., between about 10 mM and about 150 mM, between about 20 mM andabout 150 mM, between about 30 mM and about 150 mM, between about 40 mMand about 150 mM, between about 50 mM and about 150 mM, between about 60mM and about 140 mM, between about 70 mM and about 130 mM, between about80 mM and about 120 mM, between about 90 mM and about 110 mM, betweenabout 95 mM and about 105 mM, between about 30 mM and about 70 mM,between about 35 mM and about 65 mM, between about 40 mM and about 60mM, between about 45 mM and about 55 mM, between about 20 mM and about50 mM, between about 25 mM and about 45 mM, between about 30 mM andabout 40 mM, between about 30 mM and about 35 mM, between about 5 mM andabout 45 mM, between about 10 mM and about 40 mM, between about 15 mMand about 35 mM, between about 20 mM and about 30 mM, or between about20 mM and about 25 mM) methionine (or alternatively cysteine orglutathione); 5 mM to 150 mM sodium ascorbate (e.g., between about 10 mMand about 150 mM, between about 20 mM and about 150 mM, between about 30mM and about 150 mM, between about 40 mM and about 150 mM, between about50 mM and about 150 mM, between about 60 mM and about 140 mM, betweenabout 70 mM and about 130 mM, between about 80 mM and about 120 mM,between about 90 mM and about 110 mM, between about 95 mM and about 105mM, between about 10 mM and about 50 mM, between about 15 mM and about45 mM, between about 20 mM and about 40 mM, between about 25 mM andabout 35 mM, between about 30 mM and about 35 mM, between about 5 mM andabout 45 mM, between about 10 mM and about 40 mM, between about 15 mMand about 35 mM, between about 20 mM and about 30 mM, between about 20mM and about 25 mM sodium ascorbate); and 5 mM to about 150 mM (e.g.,between about 10 mM and about 150 mM, between about 20 mM and about 150mM, between about 30 mM and about 150 mM, between about 40 mM and about150 mM, between about 50 mM and about 150 mM, between about 60 mM andabout 140 mM, between about 70 mM and about 130 mM, between about 80 mMand about 120 mM, between about 90 mM and about 110 mM, between about 95mM and about 105 mM, between about 30 mM and about 70 mM, between about35 mM and about 65 mM, between about 40 mM and about 60 mM, betweenabout 45 mM and about 55 mM, between about 20 mM and about 50 mM,between about 25 mM and about 45 mM, between about 30 mM and about 40mM, between about 30 mM and about 35 mM, between about 5 mM and about 45mM, between about 10 mM and about 40 mM, between about 15 mM and about35 mM, between about 20 mM and about 30 mM, or between about 20 mM andabout 25 mM) histidine.

Non-limiting examples of any of the compositions contain (i) betweenabout 75 mM and about 125 mM (e.g., between about 80 mM and about 120mM, between about 85 mM and about 115 mM, between about 90 mM and about110 mM, or between about 95 mM and about 105 mM) mannitol (e.g., in abuffered solution, e.g., a phosphate buffer, such as 50 mM sodiumphosphate, pH 6.0); (ii) between about 75 mM and about 125 mM (e.g.,between about 80 mM and about 120 mM, between about 85 mM and about 115mM, between about 90 mM and about 110 mM, or between about 95 mM andabout 105 mM) methionine (or alternatively cysteine or glutathione)(e.g., in a buffered solution, e.g., a phosphate buffer, such as 50 mMsodium phosphate, pH 6.0); (iii) between about 75 mM and about 125 mM(e.g., between about 80 mM and about 120 mM, between about 85 mM andabout 115 mM, between about 90 mM and about 110 mM, or between about 95mM and about 105 mM) sodium ascorbate (e.g., in a buffered solution,e.g., a phosphate buffer, such as 50 mM sodium phosphate, pH 6.0); (iv)between about 75 mM and about 125 mM (e.g., between about 80 mM andabout 120 mM, between about 85 mM and about 115 mM, between about 90 mMand about 110 mM, or between about 95 mM and about 105 mM) histidine(e.g., in a buffered solution, e.g., a phosphate buffer, such as 50 mMsodium phosphate, pH 6.0); (v) between about 30 mM and about 70 mM(e.g., between about 35 mM and about 65 mM, between about 40 mM andabout 60 mM, or between about 45 mM and about 55 mM) methionine (oralternatively cysteine or glutathione) and between about 30 mM and about70 mM (e.g., between about 35 mM and about 65 mM, between about 40 mMand about 60 mM, or between about 45 mM and about 55 mM) histidine(e.g., in a buffered solution, e.g., a phosphate buffer, such as 50 mMsodium phosphate, pH 6.0); (vi) between about 10 mM and about 50 mM(e.g., between about 15 mM and about 45 mM, between about 20 mM andabout 40 mM, between about 25 mM to about 35 mM, or between about 30 mMand about 35 mM) methionine (or alternatively cysteine or glutathione),between about 10 mM and about 50 mM (e.g., between about 15 mM and about45 mM, between about 20 mM and about 40 mM, between about 25 mM to about35 mM, or between about 30 mM and about 35 mM) histidine, and betweenabout 10 mM and about 50 mM (e.g., between about 15 mM and about 45 mM,between about 20 mM and about 40 mM, between about 25 mM to about 35 mM,or between about 30 mM to about 35 mM) sodium ascorbate (e.g., in abuffered solution, e.g., a phosphate buffer, such as 50 mM sodiumphosphate, pH 6.0); or (vii) between about 5 mM and about 45 mM (e.g.,between about 10 mM and about 40 mM, between about 15 mM and about 35mM, between about 20 mM and about 30 mM, or between about 23 mM andabout 27 mM) sodium ascorbate, between about 5 mM and about 45 mM (e.g.,between about 10 mM and about 40 mM, between about 15 mM and about 35mM, between about 20 mM and about 30 mM, or between about 23 mM andabout 27 mM) methionine (or alternatively cysteine or glutathione),between about 5 mM and about 45 mM (e.g., between about 10 mM and about40 mM, between about 15 mM and about 35 mM, between about 20 mM andabout 30 mM, or between about 23 mM and about 27 mM) mannitol, andbetween about 5 mM and about 45 mM (e.g., between about 10 mM and about40 mM, between about 15 mM and about 35 mM, between about 20 mM andabout 30 mM, or between about 23 mM and about 27 mM) histidine (e.g., ina buffered solution, e.g., a phosphate buffer, such as 50 mM sodiumphosphate, pH 6.0).

Non-limiting doses of gamma-irradiation sufficient to reduce bioburdenof any of the compositions provided herein are described below.Additional doses of gamma-irradiation sufficient to reduce bioburden ofany of the compositions provided herein are known in the art. Forexample, any of the compositions described herein can begamma-irradiated at any of the doses, at any of the rates ofgamma-irradiation, and/or at any of the temperatures for performinggamma-irradiation described herein (in any combination). The bioburdenof a composition can be determined, e.g., by taking a sample from thecomposition that would contain self-replicating biologicalcontaminant(s) present in the composition, e.g., by stomaching,ultrasonicating, shaking, vortex mixing, flushing, blending, orswabbing, and qualitating or quantifying the level of self-replicatingbiological contaminant(s) present in the sample (e.g., by placing thesample in a growth medium that would allow the biological contaminant toself-replicate, e.g., plating the sample on a petri dish, or running thesample through a membrane).

The amount of the at least one antioxidant agent and/or chelatorsufficient to ameliorate the loss of binding capacity of thechromatography resin upon treatment with a dose of gamma-irradiationsufficient to reduce bioburden of the composition can be determined,e.g., using methods described in the Examples. For example, the level ofreduction in the binding capacity of a chromatography resin treated withgamma-irradiation in the presence of an amount of the at least oneantioxidant agent and/or chelator can be compared to the level ofreduction in the binding capacity of the chromatography resin treatedwith the same dose of gamma-irradation in the absence of the at leastone antioxidant agent and/or chelator, where a decrease in the level ofreduction in the binding capacity of the chromatography resingamma-irradiated in the presence of the at least one antioxidant agentand/or chelator as compared to the chromatography resin gamma-irradiatedin the absence of the at least one antioxidant agent and/or chelator,indicates that the at least one antioxidant agent and/or chelator waspresent in an amount sufficient to ameliorate the loss of bindingcapacity of the chromatography resin upon treatment withgamma-irradiation. Exemplary methods for determining the bindingcapacity of a chromatography resin are described in the Examples.Additional examples of methods for determining the binding capacity of achromatography resin are known in the art.

Methods of Reducing Bioburden of a Chromatography Resin

Provided herein are methods of reducing bioburden of a chromatographyresin. These methods include a step of exposing a container including acomposition including a chromatography resin and at least oneantioxidant agent and/or chelator to a dose of gamma-irradiationsufficient to reduce the bioburden of the container and thechromatography resin, where the at least one antioxidant agent and/orchelator are present in an amount sufficient to ameliorate the loss ofbinding capacity of the chromatography resin after (or upon) exposure tothe dose of gamma-irradiation.

Also provided are methods of reducing bioburden of a chromatographyresin that include exposing a container including a compositionincluding a chromatography resin (and optionally at least oneantioxidant agent and/or chelator in an amount sufficient to ameliorateloss of binding capacity of the chromatography resin after/upon exposureto gamma-irradiation) to gamma-irradiation at a rate of between about0.1 kGy/hour to about 6 kGy/hour (e.g., between about 0.1 kGy/hour toabout 5.5 kGy/hour, between about 0.1 kGy/hour to about 5.0 kGy/hour,between about 0.1 kGy/hour to about 4.5 kGy/hour, between about 0.1kGy/hour to about 4.0 kGy/hour, between about 0.1 kGy/hour to about 3.5kGy/hour, between about 0.1 kGy/hour to about 3.0 kGy/hour, betweenabout 0.1 kGy/hour to about 2.5 kGy/hour, between about 0.1 kGy/hour toabout 2.0 kGy/hour, between about 0.1 kGy/hour to about 1.5 kGy/hour,between about 0.1 kGy/hour to about 1.0 kGy/hour, between about 0.5kGy/hour to about 6 kGy/hour, between about 0.5 kGy/hour to about 5.5kGy/hour, between about 0.5 kGy/hour to about 5.0 kGy/hour, betweenabout 0.5 kGy/hour to about 4.5 kGy/hour, between about 0.5 kGy/hour toabout 4.0 kGy/hour, between about 0.5 kGy/hour to about 3.5 kGy/hour,between about 0.5 kGy/hour to about 3.0 kGy/hour, between about 0.5kGy/hour to about 2.5 kGy/hour, between about 0.5 kGy/hour to about 2.0kGy/hour) and/or at a temperature between about 4° C. to about 25° C.(e.g., between about 4° C. to about 20° C., between about 4° C. to about15° C., between about 4° C. to about 10° C., between about 10° C. toabout 25° C., between about 10° C. to about 20° C., between about 10° C.to about 15° C., or between about 15° C. to about 25° C.) for a dose ofgamma-irradiation sufficient to reduce the bioburden of the containerand the chromatography resin. In the methods described in thisparagraph, the level of binding capacity of the gamma-irradiatedchromagraphy resin produced by these methods is greater than the levelof binding capacity of a gamma-irradiated chromagraphy resingamma-irradiated at one or both of a rate of greater than 6.1 kGy/hourand/or at a temperature greater than 25° C.

A chromatography resin can be exposed to gamma-irradiation using methodsknown in the art. For example, an isotope such as Cobalt-60 orCaesium-137 can be used as the source of gamma-rays. The chromatographyresin can be exposed to gamma-irradiation at a temperature of aboutbetween about −25° C. and about 0° C., inclusive, or between about 0° C.and about 25° C., inclusive. The chromagraphy resin can be exposed to adose of gamma-irradiation of between about 0.1 kGy to about 100 kGy,between about 1 kGy to about 100 kGy, between about 1 kGy to about 90kGy, between about 1 kGy to about 80 kGy, between about 1 kGy to about70 kGy, between about 1 kGy to about 65 kGy, between about 5 kGy toabout 65 kGy, between about 10 kGy to about 60 kGy, between about 10 kGyto about 55 kGy, between about 10 kGy to about 50 kGy, between about 10kGy to about 45 kGy, between about 10 kGy to about 40 kGy, between about10 kGy to about 35 kGy, between about 10 kGy to about 30 kGy, betweenabout 15 kGy to about 50 kGy, between about 15 kGy to about 45 kGy,between about 15 kGy to about 40 kGy, between about 15 kGy to about 35kGy, between about 20 kGy to about 30 kGy, or between about 23 kGy toabout 27 kGy. The chromatography resin can be exposed togamma-irradiation in a dose sufficient to result in a sterilityassurance level of the chromatography resin of about or less than1×10⁻⁶, about or less than 1×10⁻⁷, about or less than 10×10⁻⁸, about orless than 1×10⁻¹¹, or about or less than 1×10⁻¹², or between about1×10⁻⁶ and about 1×10⁻¹², between about 1×10⁻⁶ and about 1×10⁻¹¹,between about 1×10⁻⁶ and about 1×10⁻¹⁰, between about 1×10⁻⁶ and about1×10⁻⁹, between about 1×10⁻⁶ and about 1×10⁻⁸, between 1×10⁻⁶ and about1×10⁻⁷ between about 1×10⁻⁷ and about 1×10⁻¹², between about 1×10⁻⁷ andabout 1×10⁻¹¹, between about 1×10⁻⁷ and about 1×10⁻¹⁰, between about1×10⁻⁷ and about 1×10⁻⁹, between about 1×10⁻⁷ and about 1×10⁻⁸, betweenabout 1×10⁻⁸ and about 1×10⁻¹², between about 1×10⁻⁸ and about 1×10⁻¹¹,between about 1×10⁻⁸ and about 1×10⁻¹⁰, or between about 1×10⁻⁸ andabout 1×10⁻⁹.

A dose of gamma-irradiation sufficient to reduce the bioburden of achromatography resin can be determined using methods known in the art.For example, the bioburden level of a chromatography resin treated witha dose of gamma-irradiation can be compared to the bioburden level of anuntreated (e.g., a control, non-gamma-irradiated) chromatography resin,and a decrease in the level of bioburden in the gamma-irradiatedchromatography resin as compared to the untreated chromatography resinindicates that the dose of gamma-irradiation is sufficient to reduce thebioburden of a chromatography resin. Exemplary methods for determiningthe level of bioburden in a composition (e.g., a chromatography resin)are described herein. Additional methods for determining the level ofbioburden in a composition (e.g., a chromatography resin) are known inthe art.

The chromatography resin in any of these methods can be an anionicexchange chromatography resin, cationic exchange chromatography resin,size exclusion chromatography resin, hydrophobic interactionchromatography resin, or an affinity chromatography resin, or anycombination thereof. Non-limiting examples of an affinity chromatographyresin can include a protein or peptide ligand (e.g., between about 5amino acids to about 100 amino acids, between about 5 amino acids toabout 90 amino acids, between about 5 amino acids and about 80 aminoacids, between about 5 amino acids and about 70 amino acids, betweenabout 5 amino acids and about 60 amino acids, between about 5 aminoacids and about 50 amino acids, between about 5 amino acids and about 40amino acids, between about 5 amino acids and about 30 amino acids,between about 5 amino acids and about 25 amino acids, or between about 5amino acids and about 20 amino acids), a small molecule substrate orcofactor of an enzyme, an aptamer, an inhibitor (e.g., competitiveprotein inhibitor) or a metal. In some embodiments, the affinitychromatography resin includes a protein ligand (e.g., protein A).Additional examples of affinity chromatography resin include a cofactorligand, a substrate ligand, a metal ligand, a product ligand, or anaptamer ligand. In some examples, the chromatography resin is a biomodalchromatography resin (e.g., anionic exchange chromatography resin andhydrophobic interaction chromatography resin). The chromatography resincan be an anionic exchange chromatography resin (e.g., an anionicexchange chromatography resin including N-benzyl-N-methyl-ethanolaminegroups).

The container that includes a chromatography resin can be a plasticcontainer (e.g., a cylindrical tube, a sealed or clamped box, or asealed bag). Non-limiting examples of containers used in these methodsinclude a storage vessel or a chromatography column. For example, thecomposition including the chromatography resin and the at least oneantioxidant agent and/or chelator can be present in a sealed container(e.g., a slurry in a sealed container or a packed chromatography resinin a sealed container (e.g., chromatography column)) A container used inthe methods described herein can be a disposable chromatography column.In some embodiments, the container used in the methods described hereinis a disposable chromatography column placed in a blister pack. Thecontainer (e.g., storage vessel or chromatography column) can have aninternal total volume of between about 1 mL to about 1 L (e.g., betweenabout 1 mL and about 900 mL, between about 1 mL and about 800 mL,between about 1 mL and about 700 mL, between about 1 mL and about 600mL, between about 1 mL and about 500 mL, between about 1 mL and about450 mL, between about 1 mL and about 400 mL, between about 1 mL andabout 350 mL, between about 1 mL and about 300 mL, between about 1 mLand about 250 mL, between about 1 mL and about 200 mL, between about 1mL and about 150 mL, between about 1 mL and about 100 mL, between about1 mL and about 75 mL, between about 1 mL and about 50 mL, between about1 mL and about 40 mL, between about 1 mL and about 30 mL, or betweenabout 1 mL and about 20 mL).

The composition containing a chromatography resin and the at least oneantioxidant agent and/or chelator included in the container can bepresent in a solid mixture (e.g., a dry or wetted solid mixture). Forexample, the container can include a slurry of a sedimentedchromatography resin in a liquid (e.g., buffered solution) containingthe at least one antioxidant agent and/or chelator. In some embodiments,the container can include a packed chromatography resin. For example,the container including the composition including the chromatographyresin and at least one antioxidant agent and/or chromatography resin isa packed chromatography column (e.g., where the resin is packed in aliquid (e.g., a buffered solution) containing the at least oneantioxidant agent and/or chromatography resin). Some embodimentsinclude, prior to exposing, disposing the composition containing thechromatography resin and the at least one antioxidant agent and/orchelator into the container.

Any of the antioxidant agents and/or chelators described herein can beused in any combination, using any combination of the exemplaryconcentrations described herein. For example, the composition caninclude at least one antioxidant agent selected from: reducedglutathione, reduced thioredoxin, reduced cysteine, a carotenoid,melatonin, lycopene, tocopherol, reduced ubiquinone, ascorbate,bilirubin, uric acid, lipoic acid, a flavonoid, a phenolpropanoid acid,lidocaine, naringenin, fullerene, glucose, mannitol,4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, and dimethylmethoxychromanol, and/or at least one chelator selected from: EDTA, DMPS, DMSA,metallothionin, and desferroxamine.

Exemplary methods for determining/identifying amount(s) of the at leastone antioxidant agent and/or chelator sufficient to ameliorate the lossof binding capacity of the chromatography resin upon treatment with adose of gamma-irradiation sufficient to reduce bioburden of thecomposition are described herein. Additional methods fordetermining/identifying amount(s) of the at least one antioxidant agentand/or chelator sufficient to ameliorate the loss of binding capacity ofthe chromatography resin upon treatment with a dose of gamma-irradiationsufficient to reduce bioburden of the composition are known in the art.

Also provided herein are a reduced bioburden chromatography resinproduced by any of the methods described herein (e.g., a reducedbioburden chromatography resin provided in a storage container, e.g., asealed storage container). The reduced bioburden chromatography resingenerated using any of the methods described herein can have a sterilityassurance level of about or less than 1×10⁻⁶, about or less than 1×10⁻⁷,about or less than 10×10⁻⁸, about or less than 1×10⁻¹¹, or about or lessthan 1×10⁻¹², or between about 1×10⁻⁶ and about 1×10⁻¹², between about1×10⁻⁶ and about 1×10⁻¹¹, between about 1×10⁻⁶ and about 1×10⁻¹⁰,between about 1×10⁻⁶ and about 1×10⁻⁹, between about 1×10⁻⁶ and about1×10⁻⁸, between 1×10⁻⁶ and about 1×10⁻⁷, between about 1×10⁻⁷ and about1×10⁻¹², between about 1×10⁻⁷ and about 1×10⁻¹¹, between about 1×10⁻⁷and about 1×10⁻¹⁰, between about 1×10⁻⁷ and about 1×10⁻⁹, between about1×10⁻⁷ and about 1×10⁻⁸, between about 1×10⁻⁸ and about 1×10⁻¹², betweenabout 1×10⁻⁸ and about 1×10⁻¹¹, between about 1×10⁻⁸ and about 1×10⁻¹⁰,or between about 1×10⁻⁸ and about 1×10⁻⁹. A reduced bioburdenchromatography resin produced by any of the methods described herein canhave a binding capacity that is at least 74% (e.g., at least 76%, atleast 78%, at least 80%, at least 82%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) or betweenabout 74% and 95%, between about 74% and about 95%, between about 76%and about 95%, at least about 78% and about 95%, between about 80% andabout 95%, or between about 74% and about 90%, between about 76% andabout 90%, between about 78% and about 90%, or between about 80% andabout 90% of the binding capacity of the same chromatography resin thathas not been treated to reduce its bioburden (e.g., has not beengamma-irradiated), when the same protein is used to test the bindingcapacity of both the chromatography resin produced by the methodsdescribed herein and the control, untreated chromatography resin.

Methods of Making a Reduced Bioburden Packed Chromatography Column

Also provided herein are methods of making a reduced packedchromatography column that includes providing a reduced bioburdenchromatography resin produced by any of the methods described herein andpacking the chromatography resin into a reduced bioburden column in anaseptic or reduced bioburden environment. In some embodiments, a reducedbioburden packed chromatography column can be produced by exposing acolumn including a packed chromatography resin and at least oneantioxidant agent and/or chelator to a dose of gamma-irradiationsufficient to reduce the bioburden of the column and the packedchromatography resin, where the at least one antioxidant agent and/orchelator is present in an amount sufficient to ameliorate the loss ofbinding capacity of the packed chromatography resin after exposure tothe dose of gamma-irradiation.

Also provided are reduced bioburden packed chromatography column(s)produced by the methods described herein. Any of the reduced bioburdenpacked chromatography column(s) produced by the methods described hereincan have a sterility assurance level of about or less than 1×10⁻⁶, aboutor less than 1×10⁻⁷, about or less than 10×10⁻⁸, about or less than1×10⁻¹¹, or about or less than 1×10⁻¹², or between about 1×10⁻⁶ andabout 1×10⁻¹², between about 1×10⁻⁶ and about 1×10⁻¹¹, between about1×10⁻⁶ and about 1×10⁻¹⁰, between about 1×10⁻⁶ and about 1×10⁻⁹, betweenabout 1×10⁻⁶ and about 1×10⁻⁸, between 1×10⁻⁶ and about 1×10⁻⁷, betweenabout 1×10⁻⁷ and about 1×10⁻¹², between about 1×10⁻⁷ and about 1×10⁻¹¹,between about 1×10⁻⁷ and about 1×10⁻¹⁰, between about 1×10⁻⁷ and about1×10⁻⁹, between about 1×10⁻⁷ and about 1×10⁻⁸, between about 1×10⁻⁸ andabout 1×10⁻¹², between about 1×10⁻⁸ and about 1×10⁻¹¹, between about1×10⁻⁸ and about 1×10⁻¹⁰, or between about 1×10⁻⁸ and about 1×10⁻⁹. Anyof the reduced bioburden packed chromatography column(s) produced by themethods described herein can contain at least one chromatography resinselected from the group of: anionic exchange chromatography resin,cationic exchange chromatography resin, affinity chromatography resin(e.g., any of the affinity chromatography resins described herein orknown in the art), hydrophilic interaction chromatography resin, andsize exclusion chromatography resin. For example, any of the reducedbioburden packed chromatography columns described herein can include anaffinity chromatography resin including a protein ligand (e.g., proteinA). A reduced bioburden packed chromatography column described hereincan include an anionic exchange chromatography resin (e.g., an anionicexchange chromatography resin including N-benzyl-N-methyl-ethanolaminegroups).

Methods of Performing Reduced Bioburden Chromatography

The methods described herein include the use of a reduced bioburdenpacked chromatography column provided herein and the processes describedherein include the use of one or two MCCSs that include at least onereduced bioburden packed chromatography column provided herein. Thegamma-irradiated chromatography resin can be any type of resin describedherein (or any type of chromatography resin known in the art).

The reduced bioburden packed chromatography column can be prepared usingany of the methods described herein. For example, the reduced bioburdenpacked chromatography column can be produced by packing a chromatographycolumn with a composition including a chromatography resin(s) and atleast one antioxidant and/or chelator (e.g., any of such compositionsdescribed herein), and exposing the packed column to gamma-irradiation(e.g., using any of the exposures and conditions described herein). Inother examples, the reduced bioburden packed chromatography column canbe produced by exposing a container including a chromatography resin andat least one antioxidant agent and/or chelator to a dose ofgamma-irradiation and packing a chromatography column with resultingreduced bioburden chromatography resin. In such methods, thechromatography resin (present in the container during exposure togamma-irradiation) can be present as a slurry in the container, and thechromatography column is packed in a reduced bioburden hood. In othermethods, the chromatography resin present in the container with the atleast one antioxidant agent and/or chelator can be exposed togamma-irradiation as a solid mixture in the container, and a slurry ofthe resulting reduced bioburden chromatography resin can be preparedusing a reduced bioburden buffer (e.g., prepared in a reduced bioburdenhood), and the resulting slurry used to pack a chromatography column ina reduced bioburden hood. In some of these examples, the chromatographycolumn, prior to packing, can be treated to reduce the bioburden (e.g.,autoclaved, gamma-irradiated, or exposure to ethylene oxide).

The reduced bioburden packed chromatography column used in any of themethods described herein can have a sterility assurance level (SAL) ofbetween about 1×10⁻³ and about 1×10⁻¹², between about 1×10⁻⁴ and about1×10⁻¹², between 1×10⁻⁵ and about 1×10⁻¹¹, between about 1×10⁻⁵ andabout 1×10⁻¹⁰, between about 1×10⁻⁵ and about 1×10⁻⁹, between about1×10⁻⁶ and about 1×10⁻⁹, or between about 1×10⁻⁶ and about 1×10⁻⁸,inclusive.

Reduced Bioburden Buffers

The methods and processes described herein can be performed using one ormore reduced bioburden buffers. As can be appreciated in the art, areduced bioburden buffer can be any type of buffer used in a cycle ofchromatography (e.g., a buffer used in any of the steps in a cycle ofchromatography or in any of the unit operations described herein).Exemplary methods for reducing the bioburden of a buffer includefiltration (0.2 μm-pore size filtration), autoclaving, andgamma-irradiation. Additional methods for reducing the bioburden of abuffer are known in the art. A reduced bioburden buffer can have asterility assurance level of between about 1×10⁻³ and about 1×10⁻¹²,between about 1×10⁻⁴ and about 1×10⁻¹², between 1×10⁻⁵ and about1×10⁻¹¹, between about 1×10⁻⁵ and about 1×10⁻¹⁰, between about 1×10⁻⁵and about 1×10⁻⁹, between about 1×10⁻⁶ and about 1×10⁻⁹, or betweenabout 1×10⁻⁶ and about 1×10⁻⁸, inclusive.

Recombinant Therapeutic Proteins

A recombinant protein as described herein can be a recombinanttherapeutic protein. Non-limiting examples of recombinant therapeuticproteins that can be produced by the methods provided herein includeimmunoglobulins (including light and heavy chain immunoglobulins,antibodies, or antibody fragments (e.g., any of the antibody fragmentsdescribed herein), enzymes (e.g., a galactosidase (e.g., analpha-galactosidase), Myozyme®, or Cerezyme®), proteins (e.g., humanerythropoietin, tumor necrosis factor (TNF), or an interferon alpha orbeta), or immunogenic or antigenic proteins or protein fragments (e.g.,proteins for use in a vaccine). Non-limiting examples of recombinanttherapeutic enzymes that can be used to treat a variety of lysosomalstorage diseases are shown in FIG. 10. The recombinant therapeuticprotein can be an engineered antigen-binding polypeptide that includesat least one multifunctional recombinant protein scaffold (see, e.g.,the recombinant antigen-binding proteins described in Gebauer et al.,Current Opin. Chem. Biol. 13:245-255, 2009; and U.S. Patent ApplicationPublication No. 2012/0164066 (herein incorporated by reference in itsentirety)). Non-limiting examples of recombinant therapeutic proteinsthat are antibodies include: panitumumab, omalizumab, abagovomab,abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab,alacizumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab,anatumomab, apolizumab, atinumab, tocilizumab, basilizimab, bectumomab,belimumab, bevacizumab, biciromab, canakinumab, cetuximab, daclizumab,densumab, eculizumab, edrecolomab, efalizumab, efungumab, ertumaxomab,etaracizumab, golimumab, infliximab, natalizumab, palivizumab,panitumumab, pertuzumab, ranibizumab, rituximab, tocilizumab, andtrastuzumab. Additional examples of recombinant therapeutic antibodiesthat can be produced by the methods described herein are known in theart. Additional non-limiting examples of recombinant therapeuticproteins that can be produced/purified by the present methods include:alglucosidase alfa, laronidase, abatacept, galsulfase, lutropin alfa,antihemophilic factor, agalsidase beta, interferon beta-1a, darbepoetinalfa, tenecteplase, etanercept, coagulation factor IX, folliclestimulating hormone, interferon beta-1a, imiglucerase, dornase alfa,epoetin alfa, and alteplase.

A secreted, soluble recombinant therapeutic protein can be recoveredfrom the liquid culture medium (e.g., a first and/or second liquidculture medium) by removing or otherwise physically separating theliquid culture medium from the cells (e.g., mammalian cells). A varietyof different methods for removing liquid culture medium from cells(e.g., mammalian cells) are known in the art, including, for example,centrifugation, filtration, pipetting, and/or aspiration. The secretedrecombinant therapeutic protein can then be recovered and furtherpurified from the liquid culture medium using a variety of biochemicaltechniques including various types of chromatography (e.g., affinitychromatography, molecular sieve chromatography, cation exchangechromatography, anion exchange chromatography, or hydrophobicinteraction chromatography, or any combination thereof) and/orfiltration (e.g., molecular weight cut-off filtration).

Cycle of Chromatography

As is well-known in the art, the steps in a cycle of chromatography candiffer depending on the chromatography resin, the buffers used toperform each step in the cycle, and the biophysical characteristics ofthe target recombinant protein (e.g., recombinant therapeutic protein).For example, an affinity chromatography column can include the steps ofloading an affinity chromatography column with a fluid including thetarget recombinant protein, washing the column to remove unwantedbiological material (e.g., contaminating proteins and/or smallmolecules), eluting the target recombinant protein bound to the column,and re-equilibrating the column. A cycle of chromatography using acationic and/or anionic exchange chromatography column, where the targetrecombinant protein binds to the chromatography resin in the loadingstep, can include the steps of loading the column with a fluid includingthe target protein, washing the column to remove unwanted biologicalmaterial, eluting the target recombinant protein bound to the column,and re-equilibrating the column. In other examples, a cycle ofchromatography using a cationic and/or anionic exchange chromatographycolumn, where unwanted biological material binds to the chromatographyresin during the loading step, while the target recombinant protein doesnot, can include the steps of loading the column with a fluid includingthe target protein, collecting the target recombinant protein in theflow-through, and reequilibrating the column. As is well-known in theart, any of the single steps in a chromatography cycle can include asingle buffer or multiple buffers (e.g., two or more buffers), and oneor more of any of the single steps in a chromatography cycle can includea buffer gradient. Any of the combination of various well-known aspectsof a single cycle of chromatography can be used in these methods in anycombination, e.g., different chromatography resin(s), flow-rate(s),buffer(s), void volume(s) of the column, bed volume(s) of the column,volume(s) of buffer used in each step, volume(s) of the fluid includingthe target protein, and the number and types of buffer(s) used in eachstep.

Methods of Performing Reduced Bioburden Column Chromatography

Provided herein are methods of performing reduced bioburdenchromatography. These methods include providing a reduced bioburdenpacked chromatography column produced using any of the methods describedherein, and performing a column chromatography using the reducedbioburden packed chromatography column. The reduced bioburden packedchromatography column can include at least one of any of thechromatography resins described herein, in any combination. For example,the chromatography resin present in the reduced bioburden packedchromatography column can be an affinity resin including a proteinligand (e.g., protein A) or can include an anionic exchangechromatography resin. The reduced bioburden packed chromatography columncan have any of the exemplary internal volumes described herein. Thereduced bioburden packed chromatography column can have any shape (e.g.,a cylinder, near cylindrical shape, or ellipsoidal shape) describedherein or known in the art. The column chromatography performed in thesemethods can be used to purify or isolate a recombinant protein (e.g.,any of the recombinant therapeutic proteins described herein or known inthe art). In some examples, the reduced bioburden packed chromatographycolumn is part of a multi-column chromatography system (MCCS), e.g., canbe part of a periodic counter current chromatography system (PCCS).

The column chromatography performed can include at least one cycle ofchromatography described herein or known in the art. For example, the atleast one cycle of chromatography can include the steps of: capturingthe recombinant protein by exposing the chromatography resin with aliquid including a recombinant protein; washing the chromatography resinby exposing the chromatography resin with a wash buffer, eluting therecombinant protein by exposing the chromatography resin with an elutionbuffer; and regenerating the chromatography resin by exposing thechromatography resin to a regeneration buffer. In some examples, theliquid including the recombinant protein is a liquid culture medium(e.g., a liquid culture medium collected from a perfusion or batchculture) or a diluted liquid culture medium (e.g., a culture mediumdiluted in buffer).

The column chromatography can be performed using a closed and integratedsystem (e.g., any of the exemplary closed and integrated systemsdescribed herein or known in the art). For example, the columnchromatography can be performed using a closed and integrated system,where the buffer is reduced bioburden buffer. As is well-known in theart, reduced bioburden buffer can be produced using a variety ofdifferent methods (e.g., prepared by filtration, by autoclaving, or heattreatment).

The column chromatography can include two or more (e.g., 3 or more, 4 ormore, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more,11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more,25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more,55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more,85 or more, 90 or more, 95 or more, or 100 or more) cycles ofchromatography. In some examples, the column chromatography is performedcontinuously over a period of at least 3 days (e.g., at least 4 days, atleast 5 days, at least 6 days, at least 7 days, at least 8 days, atleast 9 days, at least 10 days, at least 11 days, at least 12 days, atleast 13 days, at least 14 days, at least 15 days, at least 16 days, atleast 17 days, at least 18 days, at least 19 days, at least 20 days, atleast 21 days, at least 22 days, at least 23 days, at least 24 days, atleast 25 days, at least 30 days, at least 35 days, at least 40 days, atleast 45 days, at least 50 days, at least 55 days, at least 60 days, atleast 65 days, at least 70 days, at least 75 days, at least 80 days, atleast 85 days, at least 90 days, at least 95 days, or at least 100days).

In some embodiments, the chromatography resin in the reduced bioburdenpacked chromatography column has a percentage binding capacity ofbetween about 75% to about 100% (e.g., between about 76% and about 98%,between about 76% and about 96%, between about 76% and about 94%,between about 76% and about 92%, between about 76% and about 90%,between about 78% and about 100%, between about 78% and about 98%,between about 78% and about 96%, between about 78% and about 94%,between about 78% and about 92%, between about 78% and about 90%,between about 80% and about 100%, between about 80% and about 98%,between about 80% and about 96%, between about 80% and about 94%,between about 80% and about 92%, between about 80% and about 90%,between about 82% and about 100%, between about 82% and about 98%,between about 82% and about 96%, between about 82% and about 94%,between about 82% and about 92%, between about 82% and about 90%,between about 84% and about 100%, between about 84% and about 98%,between about 84% and about 96%, between about 84% and about 94%,between about 84% and about 92%, between about 84% and about 90%,between about 86% and about 100%, between about 86% and about 98%,between about 86% and about 96%, between about 86% and about 94%,between about 86% and about 92%, between about 88% and about 100%,between about 88% and about 98%, between about 88% and about 96%,between about 88% and about 94%, between about 90% and about 100%,between about 90% and about 98%, between about 90% and about 96%,between about 92% and about 100%, or between about 92% and about 98%) ascompared to the same resin not treated with gamma-irradiation (when thesame protein is used to test the binding capacity of both resins) (e.g.,assessed immediately after exposure to gamma-irradiation).

Integrated, Closed or Substantially Closed, and Continuous Processes forManufacturing of a Recombinant Protein

Provided herein are integrated, closed or substantially closed, andcontinuous processes for manufacturing a purified recombinant protein(e.g., a recombinant therapeutic protein). These processes includeproviding a liquid culture medium including a recombinant protein (e.g.,a recombinant therapeutic protein) that is substantially free of cells.

Some processes include continuously feeding the liquid culture mediuminto a multi-column chromatography system (MCCS) that includes at leastone reduced bioburden packed chromatography column provided herein,where these processes utilize reduced bioburden buffer, are integrated,and run continuously from the liquid culture medium to an eluate fromthe MCCS that is the purified recombinant protein (e.g., a therapeuticprotein drug substance).

Some processes include continuously feeding the liquid culture mediuminto a first MCCS (MCCS1), capturing the recombinant protein from theliquid culture medium using the MCCS1, producing an eluate from theMCCS1 that includes the recombinant protein and continuously feeding theeluate into a second MCCS (MCCS2), and continuously feeding therecombinant protein from the eluate into the MCCS2 and subsequentlyeluting the recombinant protein to thereby produce the purifiedrecombinant protein, where at least one column in the MCCS1 and/or theMCCS2 is a reduced bioburden packed chromatography column providedherein, the processes utilize reduced bioburden buffer, are integrated,and run continuously from the liquid culture medium to the purifiedrecombinant protein.

In some examples, each of the chromatography columns used in the MCCS,MCCS1, and/or MCCS2 is a reduced bioburden packed chromatography columnprovided herein. Some embodiments further include a step of formulatingthe purified recombinant protein into a pharmaceutical composition.

The processes described herein provide continuous and time-efficientproduction of a purified recombinant protein from a liquid culturemedium including the recombinant protein. For example, the elapsed timebetween feeding a liquid culture medium including a therapeutic proteininto the MCCS or MCCS1 and eluting the recombinant protein from the MCCSor MCCS2, respectively, can be, e.g., between about 4 hours and about 48hours, inclusive, e.g., between about 4 hours and about 40 hours,between about 4 hours and about 35 hours, between about 4 hours andabout 30 hours, between about 4 hours and about 28 hours, between about4 hours and about 26 hours, between about 4 hours and about 24 hours,between about 4 hours and about 22 hours, between about 4 hours andabout 20 hours, between about 4 hours and about 18 hours, between about4 hours and about 16 hours, between about 4 hours and about 14 hours,between about 4 hours and about 12 hours, between about 6 hours andabout 12 hours, between about 8 hours and about 12 hours, between about6 hours and about 20 hours, between about 6 hours and about 18 hours,between about 6 hours and about 14 hours, between about 8 hours andabout 16 hours, between about 8 hours and about 14 hours, between about8 hours and about 12 hours, between about 10 hours and 20 hours, betweenabout 10 hours and 18 hours, between about 10 hours and 16 hours,between about 10 hours and 14 hours, between about 12 hours and about 14hours, between about 10 hours and about 40 hours, between about 10 hoursand about 35 hours, between about 10 hours and about 30 hours, betweenabout 10 hours and about 25 hours, between about 15 hours and about 40hours, between about 15 hours and about 35 hours, between about 15 hoursand about 30 hours, between about 20 hours and about 40 hours, betweenabout 20 hours and about 35 hours, or between about 20 hours and about30 hours, inclusive. In other examples, the elapsed time between feedingthe liquid culture medium including the recombinant protein into theMCCS or MCCS1 and eluting the recombinant protein from the MCCS orMCCS2, respectively, is, e.g., greater than about 4 hours and less thanabout 40 hours, inclusive, e.g., greater than about 4 hours and lessthan about 39 hours, about 38 hours, about 37 hours, about 36 hours,about 35 hours, about 34 hours, about 33 hours, about 32 hours, about 31hours, about 30 hours, about 29 hours, about 28 hours, about 27 hours,about 26 hours, about 25 hours, about 24 hours, about 23 hours, about 22hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours,about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours,about 8 hours, about 7 hours, about 6 hours, about 5 hours, or about 4.5hours, inclusive.

Non-limiting aspects of the MCCSs that can be used in any of theseprocesses (MCCS, MCCS1, and/or MCCS2) are described in U.S. ProvisionalPatent Application Ser. Nos. 61/775,060 and 61/856,390 (eachincorporated herein by reference).

Some exemplary processes do not utilize a holding step (e.g., do not usea reservoir (e.g., break tank) in the entire process). Others may use amaximum of 1, 2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) in theentire process. Any of the processes described herein can utilize amaximum of 1, 2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) in theentire process, where each break tank only holds a recombinant proteinfor a total time period of, e.g., between about 5 minutes and less thanabout 6 hours, inclusive, e.g., between about 5 minutes and about 5hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, orabout 30 minutes, inclusive.

Some processes utilize one, two, three, four, five, or six reservoir(s)(e.g., break tank(s)) and can have a capacity that is, e.g., between 1mL and about 300 mL, inclusive, e.g., between 1 mL and about 280 mL,about 260 mL, about 240 mL, about 220 mL, about 200 mL, about 180 mL,about 160 mL, about 140 mL, about 120 mL, about 100 mL, about 80 mL,about 60 mL, about 40 mL, about 20 mL, or about 10 mL (inclusive). Anyreservoir(s) (e.g., break tank(s)) used (in any of the processesdescribed herein) to hold fluid before it is fed into the MCCS or MCCS1can have a capacity that is, e.g., between 1 mL and about 100%,inclusive, e.g., between 1 mL and about 90%, about 80%, about 70%, about60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 5%,inclusive, of the loading volume of the first column of the MCCS orMCCS1. A reservoir(s) (e.g., break tanks(s)) can be used to hold eluatefrom MCCS1 before it enters into the MCCS2 and can have a capacity thatis, e.g., between 1 mL and about 100%, inclusive, e.g., between 1 mL andabout 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about30%, about 20%, about 10%, or about 5%, inclusive, of the loading volumeof the first column of the MCCS2.

Various additional aspects of these processes are described in detailbelow and can be used in any combination in the processes providedherein without limitation. Exemplary aspects of the provided processesare described below; however, one skilled in the art will appreciatethat additional steps can be added to the processes described herein andother materials can be used to perform any of the steps of the processesdescribed herein.

Liquid Culture Medium

Liquid culture medium that includes a recombinant protein (e.g.,recombinant therapeutic protein) that is substantially free of cells canbe derived from any source. For example, the liquid culture medium canbe obtained from a recombinant cell culture (e.g., a recombinantbacterial, yeast, or mammalian cell culture). The liquid culture mediumcan be obtained from a fed-batch cell (e.g., mammalian cell) culture(e.g., a fed-batch bioreactor including a culture of mammalian cellsthat secrete the recombinant protein) or a perfusion cell (e.g.,mammalian cell) culture (e.g., a perfusion bioreactor including aculture of mammalian cells that secrete the recombinant protein). Theliquid culture medium can also be a clarified liquid culture medium froma culture of bacterial or yeast cells that secrete the recombinantprotein.

Liquid culture medium obtained from a recombinant cell culture can befiltered or clarified to obtain a liquid culture medium that issubstantially free of cells and/or viruses. Methods for filtering orclarifying a liquid culture medium in order to remove cells are known inthe art (e.g., 0.2-μm filtration and filtration using an AlternatingTangential Flow (ATF™) system). Recombinant cells can also be removedfrom liquid culture medium using centrifugation and removing thesupernatant that is liquid culture medium that is substantially free ofcells, or by allowing the cells to settle to the gravitational bottom ofa container (e.g., bioreactor) including the liquid culture medium, andremoving the liquid culture medium (the liquid culture medium that issubstantially free of cells) that is distant from the settledrecombinant cells.

The liquid culture medium can be obtained from a culture of recombinantcells (e.g., recombinant bacteria, yeast, or mammalian cells) producingany of the recombinant proteins (e.g., recombinant therapeutic proteins)described herein or known in the art. Some examples of any of theprocesses described herein can further include a step of culturingrecombinant cells (e.g., recombinant bacteria, yeast, or mammaliancells) that produce the recombinant protein (e.g., recombinanttherapeutic protein).

The liquid culture medium can be any of the types of liquid culturemedium described herein or known in the art. For example, the liquidculture medium can be selected from the group of: animal-derivedcomponent-free liquid culture medium, serum-free liquid culture medium,serum-containing liquid culture medium, chemically-defined liquidculture medium, and protein-free liquid culture medium. In any of theprocesses described herein, a liquid culture medium obtained from aculture can be diluted by addition of a second fluid (e.g., a buffer)before it is fed into the MCCS or MCCS1.

The liquid culture medium including a recombinant protein that issubstantially free of cells can be stored (e.g., at a temperature belowabout 15° C. (e.g., below about 10° C., below about 4° C., below about0° C., below about −20° C., below about −50° C., below about −70 C.°, orbelow about −80° C.) for at least 1 day (e.g., at least about 2 days, atleast about 5 days, at least about 10 days, at least about 15 days, atleast about 20 days, or at least about 30 days) prior to feeding theliquid culture medium into the MCCS or MCCS1. Alternatively, in someexamples the liquid culture medium is fed into the MCCS or MCCS1directly from a bioreactor (e.g., fed into the MCCS or MCCS1 directlyfrom the bioreactor after a filtering or clarification step).

Multi-Column Chromatography Systems

The processes described herein include the use of a MCCS or two or more(e.g., two, three, four, five, or six) multi-column chromatographysystems (MCCSs) (e.g., an MCCS1 and MCCS2). A MCCS can include two ormore chromatography columns, two or more chromatographic membranes, or acombination of at least one chromatography column and at least onechromatographic membrane. In non-limiting examples, a MCCS (e.g., MCCS,MCCS1, and/or MCCS2 in any of the processes herein) can include fourchromatographic columns, three chromatographic columns and achromatographic membrane, three chromatographic columns, twochromatographic columns, two chromatographic membranes, and twochromatographic columns and one chromatographic membrane. Additionalexamples of combinations of chromatography columns and/orchromatographic membranes can be envisioned for use in an MCCS (e.g.,MCCS, MCCS1, and/or MCCS2 in any of the processes described herein) byone skilled in the art without limitation. The individual chromatographycolumns and/or chromatographic membranes present in a MCCS can beidentical (e.g., have the same shape, volume, resin, capture mechanism,and unit operation), or can be different (e.g., have one or more of adifferent shape, volume, resin, capture mechanism, and/or unitoperation). The individual chromatography column(s) and/orchromatographic membrane(s) present in a MCCS (e.g., MCCS, MCCS1, and/orMCCS2 in any of the processes described herein) can perform the sameunit operation (e.g., the unit operation of capturing, purifying, orpolishing) or different unit operations (e.g., different unit operationsselected from, e.g., the group of capturing, purifying, polishing,inactivating viruses, adjusting the ionic concentration and/or pH of afluid including the recombinant protein, and filtering). For example, inexamples of the processes described herein, at least one chromatographycolumn and/or chromatographic membrane in the MCCS or MCCS1 performs theunit operation of capturing the recombinant protein.

The one or more chromatography column(s) that can be present in an MCCS(e.g., present in the MCCS, MCCS1, and/or MCCS2) can have a resin volumeof, e.g., between about 1 mL and about 2 mL, about 5 mL, about 10 mL,about 15 mL, about 20 mL, about 25 mL, about 30 mL, about 35 mL, about40 mL, about 45 mL, about 50 mL, about 55 mL, about 60 mL, about 65 mL,about 70 mL, about 75 mL, about 80 mL, about 85 mL, about 90 mL, about95 mL, or about 100 mL, inclusive. The one or more chromatographycolumn(s) that can be present in an MCCS (e.g., present in the MCCS,MCCS1, and/or MCCS2) can have a resin volume of between about 2 mL toabout 100 mL, between about 2 mL and about 90 mL, between about 2 mL andabout 80 mL, between about 2 mL and about 70 mL, between about 2 mL andabout 60 mL, between about 2 mL and about 50 mL, between about 5 mL andabout 50 mL, between about 2 mL and about 45 mL, between about 5 mL andabout 45 mL, between about 2 mL and about 40 mL, between about 5 mL andabout 40 mL, between about 2 mL and about 35 mL, between about 5 mL andabout 35 mL, between about 2 mL and about 30 mL, between about 5 mL andabout 30 mL, between about 2 mL and about 25 mL, between about 5 mL andabout 25 mL, between about 15 mL and about 60 mL, between about 10 mLand about 60 mL, between about 10 mL and about 50 mL, and between about15 mL and about 50 mL. The one or more chromatography column(s) in anMCCS (e.g., the MCCS, MCCS1, and/or MCCS2) used in any of the processesdescribed herein can have the substantially the same resin volume or canhave different resin volumes. The flow rate used for the one or morechromatography column(s) in an MCCS (e.g., the MCCS, MCCS1, and/orMCCS2) can be, e.g., between about 0.2 mL/minute to about 25 mL/minute(e.g., between about 0.2 mL/minute to about 20 mL/minute, between about0.5 mL/minute to about 20 mL/minute, between about 0.2 mL/minute toabout 15 mL/minute, between about 0.5 mL/minute to about 15 mL/minute,between about 0.5 mL/minute to about 10 mL/minute, between about 0.5 mLminute and about 14 mL/minute, between about 1.0 mL/minute and about25.0 mL/minute, or between about 1.0 mL/minute and about 15.0mL/minute).

The one or more chromatography column (s) in an MCCS (e.g., MCCS, MCCS1,and/or MCCS2) can have substantially the same shape or can havesubstantially different shapes. For example, the one or morechromatography column(s) in an MCCS (e.g., in the MCCS, MCCS1, and/orMCCS2) can have substantially the shape of a circular cylinder or canhave substantially the same shape of an oval cylinder.

The one or more chromatographic membrane(s) that can be present in anMCCS (e.g., present in the MCCS, MCCS1, and/or MCCS2) can have a bedvolume of, e.g., between about 1 mL to about 500 mL (e.g., between about1 mL to about 475 mL, between about 1 mL to about 450 mL, between about1 mL to about 425 mL, between about 1 mL to about 400 mL, between about1 mL to about 375 mL, between about 1 mL to about 350 mL, between about1 mL to about 325 mL, between about 1 mL to about 300 mL, between about1 mL to about 275 mL, between about 1 mL to about 250 mL, between about1 mL to about 225 mL, between about 1 mL to about 200 mL, between about1 mL to about 175 mL, between about 1 mL to about 150 mL, between about1 mL to about 125 mL, between about 1 mL to about 100 mL, between about2 mL to about 100 mL, between about 5 mL to about 100 mL, between about1 mL to about 80 mL, between about 2 mL to about 80 mL, between about 5mL to about 80 mL, between about 1 mL to about 60 mL, between about 2 mLto about 60 mL, between about 5 mL to about 60 mL, between about 1 mL toabout 40 mL, between about 2 mL to about 40 mL, between about 5 mL toabout 40 mL, between about 1 mL to about 30 mL, between about 2 mL toabout 30 mL, between about 5 mL to about 30 mL, between about 1 mL andabout 25 mL, between about 2 mL and about 25 mL, between about 1 mL andabout 20 mL, between about 2 mL and about 20 mL, between about 1 mL andabout 15 mL, between about 2 mL and about 15 mL, between about 1 mL andabout 10 mL, or between about 2 mL and about 10 mL).

One or more (e.g., three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, ortwenty-four) different types of reduced bioburden buffer can be employedduring the use of the MCCS, MCCS1, and/or MCCS2 in any of the processesdescribed herein. As is known in the art, the one or more types ofreduced bioburden buffer used in the MCCS, MCCS1, and/or MCCS2 in theprocesses described herein will depend on the resin present in thechromatography column(s) and/or the chromatographic membrane(s) of theMCCS, MCCS1, and/or MCCS2, the biophysical properties of the recombinantprotein, and unit operation (e.g., any of the exemplary unit operationsdescribed herein) performed by the specific chromatography column(s)and/or chromatography membranes of the MCCS, MCCS1, and/or MCCS2. Thevolume and type of buffer employed during the use of the MCCS, MCCS1,and/or MCCS2 in any of the processes described herein can also bedetermined by one skilled in the art (e.g., discussed in more detailbelow). For example, the volume and type(s) of buffer employed duringthe use of the MCCS, MCCS1, and/or MCCS2 in any of the processesdescribed herein can be chosen in order to optimize one or more of thefollowing in the purified recombinant protein (e.g., recombinant proteindrug product): the overall yield of recombinant protein, the activity ofthe recombinant protein, the level of purity of the recombinant protein,and the removal of biological contaminants from a fluid including therecombinant protein (e.g., liquid culture medium) (e.g., absence ofactive viruses, mycobacteria, yeast, bacteria, or mammalian cells).

The MCCS, MCCS1, and/or MCCS2 can be a periodic counter currentchromatography system (PCCS). A PCCS can, e.g., include two or morechromatography columns (e.g., three columns or four columns) that areswitched in order to allow for the continuous elution of recombinantprotein from the two or more chromatography columns. A PCCS can includetwo or more chromatography columns, two or more chromatographicmembranes, or at least one chromatographic column and at least onechromatographic membrane. A column operation (cycle) generally consistsof the load, wash, eluate, and regeneration steps. In PCCSs, multiplecolumns are used to run the same steps discretely and continuously in acyclic fashion. Since the columns are operated in series, the flowthrough and wash from one column is captured by another column. Thisunique feature of PCCSs allows for loading of the resin close to itsstatic binding capacity instead of to the dynamic binding capacity, asis typical during batch mode chromatography. As a result of thecontinuous cycling and elution, fluid entering a PCCS is processedcontinuously, and the eluate including recombinant protein iscontinuously produced.

Column-switching strategy is employed to advance from one step toanother in a PCCS cycle. Examples of column switching that can be usedin a PCCS are described in U.S. Provisional Patent Application Ser. Nos.61/775,060 and 61/856,390. For example, a column switching method canemploy two automated switching operations per column: the first of whichis related to the initial product breakthrough, while the secondcoincides with column saturation. The determination of when the columnswitching operations should take place can be determined by monitoringthe recombinant protein concentration (e.g., monitoring performed by UVmonitoring) in the eluate from each chromatography column present in aPCCS. For example, column switching can be determined by any PAT toolcapable of in-line measurement of recombinant protein concentration withfeedback control. The PAT tool is capable of real-time in-linemeasurement of recombinant protein concentration with feedback control.As in known in the art, column switches can also be designed based ontime or the amount of fluid (e.g., buffer) passed through the one ormore chromatography column(s) and/or chromatographic membranes in theMCCS, MCCS1, and/or MCCS2.

In PCCSs, the residence time (RT) of the recombinant protein on the eachchromatography column and/or chromatographic membrane present in thePCCS can be decreased without increasing the column/membrane sizebecause the breakthrough from the first column/membrane can be capturedon another column/membrane in the PCCS. A continuous process system canbe designed to process liquid culture medium at any perfusion rate (D)by varying the column/membrane volume (V) and RT using the equation of:V=D*RT.

The one or more unit operations that can be performed by the MCCS or theMCC1 and/or MCCS2 used in the presently described processes include, forexample, capturing the recombinant protein, inactivating viruses presentin a fluid including the recombinant protein, purifying the recombinantprotein, polishing the recombinant protein, holding a fluid includingthe recombinant protein (e.g., using any of the exemplary break tank(s)described herein), filtering or removing particulate material and/orcells from a fluid including the recombinant protein, and adjusting theionic concentration and/or pH of a fluid including the recombinantprotein.

In some embodiments, the MCCS or the MCCS1 includes at least onechromatographic column and/or chromatographic membrane that performs theunit operation of capturing the recombinant protein. The unit operationof capturing can be performed using at least one chromatography columnand/or chromatography resin, e.g., that utilizes a capture mechanism.Non-limiting examples of capturing mechanisms include a proteinA-binding capture mechanism, an antibody- or antibody fragment-bindingcapture mechanism, a substrate-binding capture mechanism, anaptamer-binding capture mechanism, a tag-binding capture mechanism(e.g., poly-His tag-based capture mechanism), and a cofactor-bindingcapture mechanism. Capturing can also be performed using a resin thatcan be used to perform cation exchange or anion exchange chromatography,molecular sieve chromatography, or hydrophobic interactionchromatography. Non-limiting resins that can be used to capture arecombinant protein are described herein. Additional examples of resinsthat can be used to capture a recombinant protein are known in the art.

The unit operation of inactivating viruses present in a fluid includingthe recombinant protein can be performed using a MCCS, MCCS1, and/orMCCS2 (e.g., that include(s), e.g., a chromatography column, achromatography membrane, or a holding tank that is capable of incubatinga fluid including the recombinant protein at a pH of between about 3.0to 5.0 (e.g., between about 3.5 to about 4.5, between about 3.5 to about4.25, between about 3.5 to about 4.0, between about 3.5 to about 3.8, orabout 3.75) for a period of at least 30 minutes (e.g., a period ofbetween about 30 minutes to 1.5 hours, a period of between about 30minutes to 1.25 hours, a period of between about 0.75 hours to 1.25hours, or a period of about 1 hour)).

The unit operation of purifying a recombinant protein can be performedusing one or more MCCSs (e.g., a MCCS, MCCS1, and/or MCCS2) thatinclude(s), e.g., a chromatography column or chromatographic membranethat includes a resin, e.g., that utilizes a capture system.Non-limiting examples of capturing mechanisms include a proteinA-binding capture mechanism, an antibody- or antibody fragment-bindingcapture mechanism, a substrate-binding capture mechanism, anaptamer-binding capture mechanism, a tag-binding capture mechanism(e.g., poly-His tag-based capture mechanism), and a cofactor-bindingcapture mechanism. Purifying can also be performed using a resin thatcan be used to perform cation exchange or anion exchange chromatography,molecular sieve chromatography, or hydrophobic interactionchromatography. Non-limiting resins that can be used to purify arecombinant protein are described herein. Additional examples of resinsthat can be used to purify a recombinant protein are known in the art.

The unit operation of polishing a recombinant protein can be performedusing one or more MCCSs (e.g., a MCCS, MCCS1, and/or MCCS) thatinclude(s), e.g., a chromatography column or chromatographic membranethat includes a resin, e.g., that can be used to perform cationexchange, anion exchange, molecular sieve chromatography, or hydrophobicinteraction chromatography. Non-limiting resins that can be used topolish a recombinant protein are described herein. Additional examplesof resins that can be used to polish a recombinant protein are known inthe art.

The unit operation of holding a fluid including the recombinant proteincan be performed using an MCCS (e.g., a MCCS, MCCS1, and/or MCCS2) thatincludes at least one reservoir (e.g., a break tank) or a maximum of 1,2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) in the MCCS or theMCCS1 and MCCS2 combined. For example, the reservoir(s) (e.g., breaktank(s)) that can be used to achieve this unit operation can each have avolume of between about 1 mL to about 1 L (e.g., between about 1 mL toabout 800 mL, between about 1 mL to about 600 mL, between about 1 mL toabout 500 mL, between about 1 mL to about 400 mL, between about 1 mL toabout 350 mL, between about 1 mL to about 300 mL, between about 10 mLand about 250 mL, between about 10 mL and about 200 mL, between about 10mL and about 150 mL, or between about 10 mL to about 100 mL). Thereservoir(s) (e.g., break tank(s)) used in the processes describedherein can have a capacity that is, e.g., between 1 mL and about 300 mL,inclusive, e.g., between 1 mL and about 280 mL, about 260 mL, about 240mL, about 220 mL, about 200 mL, about 180 mL, about 160 mL, about 140mL, about 120 mL, about 100 mL, about 80 mL, about 60 mL, about 40 mL,about 20 mL, or about 10 mL, inclusive. Any of the reservoir(s) (e.g.,break tank(s)) used (in any of the processes described herein) to holdfluid before it enters into the MCCS or MCCS1 can have a capacity thatis, e.g., between 1 mL and about 100%, inclusive, between about 1 mL andabout 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about30%, about 20%, about 10%, or about 5%, inclusive, of the loading volumeof the first column of the MCCS or MCCS1. Any of the reservoir(s) (e.g.,break tanks(s)) used to hold a eluate from MCCS1 (including therecombinant protein) before it enters the MCCS2 can have a capacity thatis, e.g., between 1 mL and about 100%, inclusive, e.g., between about 1mL and about 90%, about 80%, about 70%, about 60%, about 50%, about 40%,about 30%, about 20%, about 10%, or about 5%, inclusive, of the loadingvolume of the first column of the MCCS2.

The reservoir(s) (e.g., break tank(s)) can each hold the fluid includingthe recombinant protein for at least 10 minutes (e.g., at least 20minutes, at least 30 minutes, at least 1 hour, at least 2 hours, atleast 4 hours, or at least 6 hours). In other examples, the reservoir(s)(e.g., break tank(s)) only holds a recombinant protein for a total timeperiod of, e.g., between about 5 minutes and less than about 6 hours,inclusive, e.g., between about 5 minutes and about 5 hours, about 4hours, about 3 hours, about 2 hours, about 1 hour, or about 30 minutes,inclusive. The reservoir(s) (e.g., break tank(s)) can be used to bothhold and refrigerate (e.g., at a temperature of less than 25° C., lessthan 15° C., or less than 10° C.) the fluid including the recombinantprotein. The reservoir can have any shape, including a circularcylinder, an oval cylinder, or an approximately rectangular sealed andnonpermeable bag.

The unit operations of filtering a fluid including the recombinantprotein can be performed using an MCCS (e.g., the MCCS, MCCS1, and/orMCCS2) that includes, e.g., a filter, or a chromatography column orchromatographic membrane that includes a molecular sieve resin. As isknown in the art, a wide variety of submicron filters (e.g., a filterwith a pore size of less than 1 μm, less than 0.5 μm, less than 0.3 μm,about 0.2 μm, less than 0.2 μm, less than 100 nm, less than 80 nm, lessthan 60 nm, less than 40 nm, less than 20 nm, or less than 10 nm) areavailable in the art that are capable of removing any precipitatedmaterial and/or cells (e.g., precipitated, unfolded protein;precipitated, unwanted host cell proteins; precipitated lipids;bacteria; yeast cells; fungal cells; mycobacteria; and/or mammaliancells). Filters having a pore size of about 0.2 μm or less than 0.2 μmare known to effectively remove bacteria from the fluid including therecombinant protein. As is known in the art, a chromatography column ora chromatographic membrane including a molecular sieve resin can also beused in an MCCS (e.g., the MCCS, MCCS1, and/or MCCS2) to perform theunit operation of filtering a fluid including a recombinant protein.

The unit operations of adjusting the ionic concentration and/or pH of afluid including the recombinant protein can be performed using a MCCS(e.g., a MCCS, a MCCS1, and/or a MCCS2) that includes and utilizes abuffer adjustment reservoir (e.g., an in-line buffer adjustmentreservoir) that adds a new buffer solution into a fluid that includesthe recombinant protein (e.g., between columns within the MCCS, MCCS1,and/or MCCS2, or after the last column in a penultimate MCCS (e.g., theMCCS1) and before the fluid including the recombinant protein is fedinto the first column of the next MCCS (e.g., the MCCS2)). As can beappreciated in the art, the in-line buffer adjustment reservoir can beany size (e.g., greater than 100 mL) and can include any bufferedsolution (e.g., a buffered solution that has one or more of: anincreased or decreased pH as compared to the fluid including therecombinant protein, an increased or decreased ionic (e.g., salt)concentration compared to the fluid including the recombinant protein,and/or an increased or decreased concentration of an agent that competeswith the recombinant protein for binding to resin present in at leastone chromatographic column or at least one chromatographic membrane inan MCCS (e.g., the MCCS, MCCS1, and/or MCCS2)).

The MCCS, MCCS1, and/or MCCS2 can perform two or more unit operations.For example, the MCCS, MCCS1, and/or MCCS2 can each perform at least thefollowing unit operations: capturing the recombinant protein andinactivating viruses present in the fluid including the recombinantprotein; capturing the recombinant protein, inactivating viruses presentin the fluid including the recombinant protein, and adjusting the ionicconcentration and/or pH of a liquid including the recombinant protein;purifying the recombinant protein and polishing the recombinant protein;purifying the recombinant protein, polishing the recombinant protein,and filtering a fluid including the recombinant protein or removingprecipitates and/or particular matter from a fluid including therecombinant protein; and purifying the recombinant protein, polishingthe recombinant protein, filtering a fluid including the recombinantprotein or removing precipitates and/or particulate matter from a fluidincluding the recombinant protein, and adjusting the ionic concentrationand/or pH of a liquid including the recombinant protein.

Capturing the Recombinant Protein

The present processes include a step of capturing the recombinantprotein using a MCCS or MCCS1. As can be appreciated in the art, theliquid culture medium including the recombinant protein can becontinuously fed onto the MCCS or MCCS1 using a variety of differentmeans. For example, the liquid culture medium can be actively pumpedinto the MCCS or MCCS1, or the liquid culture medium can be fed into theMCCS or MCCS1 using gravitational force. The liquid culture medium canbe stored in a reservoir (e.g., a holding tank) before it is fed intothe MCCS or MCCS1 or the liquid culture medium can be actively pumpedfrom a bioreactor including a culture of cells (e.g., mammalian cellsthat secrete the recombinant protein into the culture medium) into theMCCS or MCCS1.

The liquid culture medium can be fed (loaded) into the MCCS or MCCS1 ata flow rate of between about 0.2 mL/minute to about 25 mL/minute (e.g.,between about 0.2 mL/minute to about 20 mL/minute, between about 0.5mL/minute to about 20 mL/minute, between about 0.2 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 15 mL/minute, betweenabout 0.5 mL/minute to about 10 mL/minute, between about 0.5 mL minuteand about 14 mL/minute, between about 1.0 mL/minute and about 25.0mL/minute, between about 1.0 mL/minute and about 15.0 mL/minute). Theliquid culture medium including the recombinant protein can be derivedfrom any of the exemplary sources described herein or known in the art.

Some examples further include the optional step of filtering the liquidculture medium before it is fed into the MCCS or MCCS1. Any of theexemplary means of filtering a liquid culture medium or a fluidincluding the recombinant protein described herein, or any filtrationmeans known in the art, can be used to filter the liquid culture mediumincluding the recombinant protein before it is fed into the MCCS orMCCS1.

In the processes described herein, the capturing of the recombinantprotein from the liquid culture medium is performed using the MCCS orMCCS1. As can be appreciated in the art, in order to achieve the captureof the recombinant protein, at least one chromatographic column or atleast one chromatographic membrane in the MCCS or MCCS1 must include aresin that utilizes a capturing mechanism (e.g., any of the exemplarycapturing mechanisms described herein), or includes a resin capable ofperforming cation exchange, anion exchange, molecular sieve, orhydrophobic interaction chromatography. For example, if the recombinantprotein is an antibody or an antibody fragment, the capturing system canbe a protein A-binding capturing mechanism or an antigen-bindingcapturing mechanism (where the capturing antigen is specificallyrecognized by the recombinant antibody or antibody fragment). If therecombinant protein is an enzyme, the capturing mechanism can use anantibody or antibody fragment that specifically binds to the enzyme tocapture the recombinant enzyme, a substrate of the enzyme to capture therecombinant enzyme, a cofactor of the enzyme to capture the recombinantenzyme, or, if the recombinant enzyme includes a tag, a protein, metalchelate, or antibody (or antibody fragment) that specifically binds tothe tag present in the recombinant enzyme. Non-limiting resins that canbe used to capture a recombinant protein are described herein andadditional resins that can be used to capture a recombinant protein areknown in the art. One non-limiting example of resin that utilizes aprotein A-binding capture mechanism is Mab Select SuRe™ resin (GEHealthcare, Piscataway, N.J.), JSR LifeSciences Amsphere ProA JWT203(Sunnyvale, Calif.), and Kaneka KanCap A (Osaka, Japan).

Exemplary non-limiting sizes and shapes of the chromatography column(s)or chromatographic membrane(s) present in the MCCS or MCCS1 that can beused to capture the recombinant protein are described herein. The liquidculture medium fed (loaded) into the MCCS or MCCS1 can include, e.g.,between about 0.05 mg/mL to about 100 mg/mL recombinant protein (e.g.,between about 0.1 mg/mL to about 90 mg/mL, between about 0.1 mg/mL toabout 80 mg/mL, between about 0.1 mg/mL to about 70 mg/mL, between about0.1 mg/mL to about 60 mg/mL, between about 0.1 mg/mL to about 50 mg/mL,between about 0.1 mg/mL to about 40 mg/mL, between about 0.1 mg/mL toabout 30 mg/mL, between about 0.1 mg/mL to about 20 mg/mL, between 0.5mg/mL to about 20 mg/mL, between about 0.1 mg/mL to about 15 mg/mL,between about 0.5 mg/mL to about 15 mg/mL, between about 0.1 mg/mL toabout 10 mg/mL, or between about 0.5 mg/mL to about 10 mg/mL recombinantprotein). The mean time required for the recombinant protein to bind tothe resin used to perform the unit operation of capturing can be, e.g.,between about 5 seconds to about 10 minutes (e.g., between about 10seconds to about 8 minutes, between about 10 seconds to about 7 minutes,between about 10 seconds to about 6 minutes, between about 10 seconds toabout 5 minutes, between about 30 seconds to about 5 minutes, betweenabout 1 minute to about 5 minutes, between about 10 seconds to about 4minutes, between about 30 seconds to about 4 minutes, or between about 1minute to about 4 minutes).

As can be appreciated in the art, in order to capture the recombinantprotein using the chromatography column(s) or chromatographicmembrane(s) present in the MCCS or MCCS1, one must perform thesequential chromatographic steps of loading, washing, eluting, andregenerating the chromatography column(s) or chromatography membrane(s)present in the MCCS or MCCS1. Any of the exemplary flow rates, buffervolumes, and/or lengths of time allotted for each sequentialchromatographic step described herein can be used in the one or more ofthese different sequential chromatographic steps (e.g., one or more ofthe sequential chromatographic steps of loading, washing, eluting, andregenerating the chromatography column(s) or chromatography membrane(s)present in the MCCS or MCCS1 that are used for capturing the recombinantprotein). Non-limiting flow rates, buffer volumes, and/or lengths oftime allotted for each sequential chromatographic step that can be usedfor capturing chromatographic column(s) and/or chromatographicmembrane(s) in the MCCS or MCCS1 (e.g., a PCCS or PCCS1) are providedbelow. In addition, exemplary buffers that can be used in the MCCSand/or MCCS1 are described below.

The MCCS or MCCS1 including at least one chromatographic column and/orchromatographic membrane including a resin that can perform the unitoperation of capturing (e.g., any of exemplary resins that can be usedfor capturing described herein) can be loaded with the liquid culturemedium including a recombinant protein using any of loading flow rates(fed rates) described above. In some examples, a single chromatographiccolumn or single chromatographic membrane including a resin that iscapable of performing the unit operation of capturing is loaded in,e.g., between about 10 minutes to about 90 minutes (e.g., between about15 minutes and about 90 minutes, between about 20 minutes and 80minutes, between about 30 minutes and 80 minutes, between about 40minutes and about 80 minutes, between about 50 minutes and about 80minutes, and between about 60 minutes and 80 minutes). In some examples,wherein the MCCS or MCCS1 includes at least two chromatographic columnsthat include a resin that is capable of performing the unit operation ofcapturing in series, the time required to load two of thechromatographic columns in series is, e.g., between about 50 minutes toabout 180 minutes (e.g., between about 60 minutes and about 180 minutes,between about 70 minutes and about 180 minutes, between about 80 minutesand about 180 minutes, between about 90 minutes and about 180 minutes,between about 100 minutes and about 180 minutes, between about 110minutes and 150 minutes, and between about 125 minutes and about 145minutes).

Following the loading of the recombinant protein onto the at least onechromatographic column or chromatographic membrane in the MCCS or MCCS1that includes a resin that is capable of performing the unit operationof capturing, the at least one chromatographic column or chromatographicmembrane is washed with at least one washing buffer. As can beappreciated in the art, the at least one (e.g., two, three, or four)washing buffer is meant to elute all or most of proteins that are notthe recombinant protein from the at least one chromatography column orchromatographic membrane, while not disturbing the interaction of therecombinant protein with the resin.

The wash buffer can be passed through the at least one chromatographycolumn or chromatographic membrane at a flow rate of between about 0.2mL/minute to about 25 mL/minute (e.g., between about 0.2 mL/minute toabout 20 mL/minute, between about 0.5 mL/minute to about 20 mL/minute,between about 0.2 mL/minute to about 15 mL/minute, between about 0.5mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, between about 1.0mL/minute and about 15.0 mL/minute). The volume of wash buffer used(e.g., combined total volume of wash buffer used when more than one washbuffer is used) can be, e.g., between about 1× column volume (CV) toabout 15×CV (e.g., between about 1×CV to about 14×CV, about 1×CV toabout 13×CV, about 1×CV to about 12×CV, about 1×CV to about 11×CV, about2×CV to about 11×CV, about 3×CV to about 11×CV, about 4×CV to about11×CV, about 5×CV to about 11×CV, or about 5×CV to about 10×CV). Thetotal time of the washing can be, e.g., between about 2 minutes to about3 hours (e.g., between about 2 minutes to about 2.5 hours, between about2 minutes to about 2.0 hours, between about 5 minutes to about 1.5hours, between about 10 minutes to about 1.5 hours, between about 10minutes to about 1.25 hours, between about 20 minutes to about 1.25hours, or between about 30 minutes to about 1 hour).

Following the washing of the at least one chromatographic column orchromatographic membrane in the MCCS or MCCS1 that includes a resin thatis capable of performing the unit operation of capturing, therecombinant protein is eluted from the at least one chromatographiccolumn or chromatographic membrane by passing an elution buffer throughthe at least one chromatographic column or chromatographic membrane inthe MCCS or MCCS1 that includes a resin that is capable of performingthe unit operation of capturing. The elution buffer can be passedthrough the at least one chromatography column or chromatographicmembrane that includes a resin that is capable of performing the unitoperation of capturing at a flow rate of between about 0.2 mL/minute toabout 25 mL/minute (e.g., between about 0.2 mL/minute to about 20mL/minute, between about 0.5 mL/minute to about 20 mL/minute, betweenabout 0.2 mL/minute to about 15 mL/minute, between about 0.5 mL/minuteto about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL/minute and about 6.0 mL/minute, betweenabout 1.0 mL/minute and about 5.0 mg/minute, between about 0.5 mL minuteand about 14 mL/minute, between about 1.0 mL/minute and about 25.0mL/minute, or between about 1.0 mL/minute and about 15.0 mL/minute). Thevolume of elution buffer used to elute the recombinant protein from eachof the at least one chromatographic column or chromatographic membraneincluding a resin that is capable of performing the unit operation ofpurifying can be, e.g., between about 1× column volume (CV) to about15×CV (e.g., between about 1×CV to about 14×CV, about 1×CV to about13×CV, about 1×CV to about 12×CV, about 1×CV to about 11×CV, about 2×CVto about 11×CV, about 3×CV to about 11×CV, about 4×CV to about 11×CV,about 5×CV to about 11×CV, or about 5×CV to about 10×CV). The total timeof the eluting can be, e.g., between about 2 minutes to about 3 hours(e.g., between about 2 minutes to about 2.5 hours, between about 2minutes to about 2.0 hours, between about 2 minutes to about 1.5 hours,between about 2 minutes to about 1.5 hours, between about 2 minutes toabout 1.25 hours, between about 2 minutes to about 1.25 hours, betweenabout 2 minutes to about 1 hour, between about 2 minutes and about 40minutes, between about 10 minutes and about 40 minutes, or between about20 minutes and about 40 minutes). Non-limiting examples of elutionbuffers that can be used in these methods will depend on the capturemechanism and/or the recombinant protein. For example, an elution buffercan include a different concentration of salt (e.g., increased saltconcentration), a different pH (e.g., an increased or decreased saltconcentration), or a molecule that will compete with the recombinantprotein for binding to the resin that is capable of performing the unitoperation of capturing. Examples of such elution buffers for eachexemplary capture mechanism described herein are well known in the art.

Following the elution of the recombinant protein from the at least onechromatographic column or chromatographic membrane in the MCCS or theMCCS1 that includes a resin that is capable of performing the unitoperation of capturing, and before the next volume of liquid culturemedium can be loaded onto the at least one chromatographic column orchromatographic membrane, the at least one chromatography column orchromatographic membrane must be equilibrated using an regenerationbuffer. The regeneration buffer can be passed through the at least onechromatography column or chromatographic membrane that includes a resinthat is capable of performing the unit operation of capturing at a flowrate of, e.g., between about 0.2 mL/minute to about 25 mL/minute (e.g.,between about 0.2 mL/minute to about 20 mL/minute, between about 0.5mL/minute to about 20 mL/minute, between about 0.2 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 15 mL/minute, betweenabout 0.5 mL/minute to about 10 mL/minute, between about 0.5 mL/minuteand about 6.0 mL/minute, between about 1.0 mL/minute and about 5.0mg/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, between about 5.0mL/minute to about 15.0 mL/minute, or between about 1.0 mL/minute andabout 15.0 mL/minute). The volume of regeneration buffer used toequilibrate the at least one chromatography column or chromatographicmembrane that includes a resin that is capable of performing the unitoperation of capturing can be, e.g., between about 1× column volume (CV)to about 15×CV (e.g., between about 1×CV to about 14×CV, about 1×CV toabout 13×CV, about 1×CV to about 12×CV, about 1×CV to about 11×CV, about2×CV to about 11×CV, about 3×CV to about 11×CV, about 2×CV to about5×CV, about 4×CV to about 11×CV, about 5×CV to about 11×CV, or about5×CV to about 10×CV).

In some of the processes described herein, the MCCS or MCCS1 includes areservoir that holds a fluid including the recombinant protein at low pH(e.g., a pH below 4.6, below 4.4, below 4.2, below 4.0, below 3.8, below3.6, below 3.4, below 3.2, or below 3.0) for, e.g., about 1 minute to1.5 hours (e.g., about 1 hour), and inactivates the viruses present in afluid including the recombinant protein. An example of a reservoir thatcan be used to perform the unit operation of inactivating viruses is astir flask (e.g., 500-mL stir flask, e.g., a 500-mL stir flask with aprogrammed stir plate) that is capable of holding a fluid including arecombinant protein for, e.g., about 1 minute to 1.5 hours, e.g., beforethe fluid including the recombinant protein is fed into the MCCS2. Thereservoir that is used to perform the unit operation of inactivation ofviruses can be a 500-mL stir flask with a programmed stir plate (e.g., astir plate programmed to mix (e.g., periodically mix) the fluid withinthe reservoir, e.g., every 4 hours). Another example of a reservoir thatcan be used to perform the unit operation of inactivation of viruses isa plastic bag (e.g., 500-mL plastic bag) that is capable of holding afluid including a recombinant protein for, e.g., about 1 minute to 1.5hours, e.g., before the fluid including the recombinant protein is fedinto the MCCS2. In some examples, the fluid including the recombinantprotein can already have a low pH (e.g., a pH below 4.6, below 4.4,below 4.2, below 4.0, below 3.8, below 3.6, below 3.4, below 3.2, orbelow 3.0) when it is fed into the reservoir that is used to perform theunit operation of viral inactivation. As can be appreciated by thoseskilled in the art, a variety of other means can be used to perform theunit operation of inactivating viruses. For example, UV irradiation of afluid including the recombinant protein can also be used to perform theunit operation of inactivating viruses. Non-limiting examples ofreservoirs that can be used to perform the unit operation ofinactivation of viruses present in a fluid including the recombinantprotein are described herein.

The MCCS or MCCS1 can include a PCCS including four chromatographycolumns, where at least three of the four chromatography columns performthe unit operation of capturing the recombinant protein from the liquidculture medium (e.g., using an MCCS that includes any of the at leastone chromatography columns that include a resin that is capable ofperforming the unit operation of capturing (e.g., any of those describedherein)). In these examples, the fourth-column of the PCC can performthe unit operation of inactivating viruses in a fluid that includes therecombinant protein (e.g., any of the exemplary columns described hereinthat can be used to achieve viral inactivation of a fluid including therecombinant protein).

In some examples, a fluid including the recombinant protein iscontinuously eluted from the MCCS1 (e.g., PCCS1), and is continuouslyfed into the MCCS2 (e.g., PCCS2). The percent of the recombinant proteinrecovered in the eluate of the MCCS or MCCS1 (e.g., PCCS or PCCS1) canbe, e.g., at least 70%, at least 72%, at least 74%, at least 76%, atleast 78%, at least 80%, at least 82%, at least 84%, at least 86%, atleast 88%, at least 90%, at least 92%, at least 94%, at least 96%, or atleast 98%). The eluate from the MCCS1 (e.g., PCCS1) can be fed into theMCCS2 (e.g., PCCS2) using a variety of means known in the art (e.g.,tubing). The eluate of the MCCS1 (e.g., PCCS1) can be fed into the MCCS2(e.g., PCCS2) at a flow rate of, e.g., between about 0.2 mL/minute toabout 25 mL/minute (e.g., between about 0.2 mL/minute to about 20mL/minute, between about 0.5 mL/minute to about 20 mL/minute, betweenabout 0.2 mL/minute to about 15 mL/minute, between about 0.5 mL/minuteto about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL/minute and about 6.0 mL/minute, betweenabout 1.0 mL/minute and about 5.0 mg/minute, between about 0.5 mL minuteand about 14 mL/minute, between about 1.0 mL/minute and about 25.0mL/minute, between about 5.0 mL/minute to about 15.0 mL/minute, betweenabout 15 mL/minute to about 25 mL/minute, or between about 1.0 mL/minuteand about 15.0 mL/minute).

Some processes described herein can further include a step of adjustingthe ionic concentration and/or pH of the eluate from the MCCS1 (e.g.,PCCS1) before it is fed into the MCCS2 (e.g., PCCS2). As describedherein, the ionic concentration and/or pH of the eluate from the MCCS1(e.g., PCCS1) can be adjusted (before it is fed into the MCCS2) byadding a buffer to the eluate (e.g., through the use of an in-linebuffer adjustment reservoir). The buffer can be added to the eluate fromthe MCCS1 at a flow rate of, e.g., between about 0.1 mL/minute to about15 mL/minute (e.g., between about 0.1 mL/minute to about 12.5 mL/minute,between about 0.1 mL/minute to about 10.0 mL/minute, between about 0.1mL/minute to about 8.0 mL/minute, between about 0.1 mL/minute to about 6mL/minute, between about 0.1 mL/minute to 4 mL/minute, or between about0.5 mL/minute to about 5 mL/minute).

The processes described herein can further include a step of holding orstoring (and optionally also refrigerating) the eluate from the MCCS1prior to feeding the eluate from the MCCS1 into the MCCS2. As describedherein, this holding or storing step can be performed using any of thereservoirs (e.g., back-up tanks) described herein.

The processes described herein can also include a step of filtering theeluate from the MCCS1 before the eluate is fed into the MCCS2. Any ofthe exemplary filters or methods for filtration described herein can beused to filter the eluate from the MCCS1 before the eluate is fed intothe MCCS2.

Polishing and Purifying the Recombinant Protein

The MCCS, MCCS1, and/or MCCS2 can be used to perform the unit operationof purifying and polishing the recombinant protein. For example, theMCCS2 can be used to perform the operation of purifying and polishingthe recombinant protein and the eluate from the MCCS2 is a protein drugsubstance. The MCCS, MCCS1, and/or MCCS2 can include at least one (e.g.,two, three, or four) chromatography column or chromatographic membranethat can be used to perform the unit operation of purifying arecombinant protein, and at least one (e.g., two, three, or four)chromatography column or chromatographic membrane that can be used toperform the unit operation of polishing the recombinant protein.

The at least one chromatography column or chromatographic membrane thatcan be used to perform the unit operation of purifying the recombinantprotein can include a resin that utilizes a capture mechanism (e.g., anyof the capture mechanisms described herein or known in the art), or aresin that can be used to perform anion exchange, cation exchange,molecular sieve, or hydrophobic interaction chromatography. The at leastone chromatography column or chromatographic membrane that can be usedto perform the unit of operation of polishing the recombinant proteincan include a resin that can be used to perform anion exchange, cationexchange, molecular sieve, or hydrophobic interaction chromatography(e.g., any of the exemplary resins for performing anion exchange, cationexchange, molecular sieve, or hydrophobic interaction chromatographydescribed herein or known in the art).

The size, shape, and volume of the at least one chromatography column orchromatography membrane that can be used to perform the unit ofoperation of purifying the recombinant protein, and/or the size andshape of the at least one chromatographic membrane that can be used toperform the unit of operation of polishing the recombinant protein canany of combination of the exemplary sizes, shapes, and volumes ofchromatography columns or chromatographic membranes described herein. Ascan be appreciated by one skilled in the art, the step of purifying orpolishing a recombinant protein can, e.g., include the steps of loading,washing, eluting, and equilibrating the at least one chromatographycolumn or chromatographic membrane used to perform the unit of operationof purifying or polishing the recombinant protein. Typically, theelution buffer coming out of a chromatography column or chromatographicmembrane used to perform the unit operation of purifying includes therecombinant protein. Typically, the loading and/or wash buffer comingout of a chromatography column or chromatographic membrane used toperform the unit operation of polishing includes the recombinantprotein.

For example, the size of the at least one chromatography column orchromatographic membrane that can be used to perform unit operation ofpurifying the recombinant protein can have a volume of, e.g., betweenabout 2.0 mL to about 200 mL (e.g., between about 2.0 mL to about 180mL, between about 2.0 mL to about 160 mL, between about 2.0 mL to about140 mL, between about 2.0 mL to about 120 mL, between about 2.0 mL toabout 100 mL, between about 2.0 mL to about 80 mL, between about 2.0 mLto about 60 mL, between about 2.0 mL to about 40 mL, between about 5.0mL to about 40 mL, between about 2.0 mL to about 30 mL, between about5.0 mL to about 30 mL, or between about 2.0 mL to about 25 mL). The flowrate of the fluid including the recombinant protein as it is loaded ontothe at least one chromatography column or at least one chromatographicthat can be used to perform the unit operation of purifying therecombinant protein can be, e.g., between about 0.1 mL/minute to about25 mL/minute (e.g., between about 0.1 mL/minute to about 12.5 mL/minute,between about 0.1 mL/minute to about 10.0 mL/minute, between about 0.1mL/minute to about 8.0 mL/minute, between about 0.1 mL/minute to about 6mL/minute, between about 0.1 mL/minute to 4 mL/minute, between about 0.1mL/minute to about 3 mL/minute, between about 0.1 mL/minute to about 2mL/minute, or about 0.2 mL/minute to about 4 mL/minute). Theconcentration of the recombinant protein in the fluid loaded onto the atleast one chromatography column or chromatographic membrane that can beused to perform the unit operation of purifying the recombinant proteincan be, e.g., between about 0.05 mg/mL to about 100 mg/mL recombinantprotein (e.g., between about 0.1 mg/mL to about 90 mg/mL, between about0.1 mg/mL to about 80 mg/mL, between about 0.1 mg/mL to about 70 mg/mL,between about 0.1 mg/mL to about 60 mg/mL, between about 0.1 mg/mL toabout 50 mg/mL, between about 0.1 mg/mL to about 40 mg/mL, between about0.1 mg/mL to about 30 mg/mL, between about 0.1 mg/mL to about 20 mg/mL,between 0.5 mg/mL to about 20 mg/mL, between about 0.1 mg/mL to about 15mg/mL, between about 0.5 mg/mL to about 15 mg/mL, between about 0.1mg/mL to about 10 mg/mL, or between about 0.5 mg/mL to about 10 mg/mLrecombinant protein). The resin in the at least one chromatographycolumn or chromatographic membrane used to perform unit operation ofpurifying can be a resin that can be used to perform anion exchange orcation exchange chromatography. The resin in the at least onechromatography column or chromatographic membrane that is used toperform the unit operation of purifying can be a cationic exchange resin(e.g., Capto-S resin, GE Healthcare Life Sciences, Piscataway, N.J.).

Following the loading of the recombinant protein onto the at least onechromatographic column or chromatographic membrane that can be used toperform the unit operation of purifying the recombinant protein, the atleast one chromatographic column or chromatographic membrane is washedwith at least one washing buffer. As can be appreciated in the art, theat least one (e.g., two, three, or four) washing buffer is meant toelute all proteins that are not the recombinant protein from the atleast one chromatography column or chromatographic membrane, while notdisturbing the interaction of the recombinant protein with the resin orotherwise eluting the recombinant protein.

The wash buffer can be passed through the at least one chromatographycolumn or chromatographic membrane at a flow rate of between about 0.2mL/minute to about 25 mL/minute (e.g., between about 0.2 mL/minute toabout 20 mL/minute, between about 0.5 mL/minute to about 20 mL/minute,between about 0.2 mL/minute to about 15 mL/minute, between about 0.5mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, or between about 1.0mL/minute and about 15.0 mL/minute). The volume of wash buffer used(e.g., combined total volume of wash buffer used when more than one washbuffer is used) can be, e.g., between about 1× column volume (CV) toabout 15×CV (e.g., between about 1×CV to about 14×CV, about 1×CV toabout 13×CV, about 1×CV to about 12×CV, about 1×CV to about 11×CV, about2×CV to about 11×CV, about 3×CV to about 11×CV, about 4×CV to about11×CV, about 2.5×CV to about 5.0×CV, about 5×CV to about 11×CV, or about5×CV to about 10×CV). The total time of the washing can be, e.g.,between about 2 minutes to about 3 hours (e.g., between about 2 minutesto about 2.5 hours, between about 2 minutes to about 2.0 hours, betweenabout 5 minutes to about 1.5 hours, between about 10 minutes to about1.5 hours, between about 10 minutes to about 1.25 hours, between about20 minutes to about 1.25 hours, between about 30 minutes to about 1hour, between about 2 minutes and 10 minutes, between about 2 minutesand 15 minutes, or between about 2 minutes and 30 minutes).

Following the washing of the at least one chromatographic column orchromatographic membrane used to perform the unit operation of purifyingthe recombinant protein, the recombinant protein is eluted from the atleast one chromatographic column or chromatographic membrane by passingan elution buffer through the at least one chromatographic column orchromatographic membrane used to perform the unit operation of purifyingthe recombinant protein. The elution buffer can be passed through the atleast one chromatography column or chromatographic membrane that can beused to perform the unit operation of purifying the recombinant proteinat a flow rate of between about 0.2 mL/minute to about 25 mL/minute(e.g., between about 0.2 mL/minute to about 20 mL/minute, between about0.5 mL/minute to about 20 mL/minute, between about 0.2 mL/minute toabout 15 mL/minute, between about 0.5 mL/minute to about 15 mL/minute,between about 0.5 mL/minute to about 10 mL/minute, between about 0.5mL/minute and about 6.0 mL/minute, between about 1.0 mL/minute and about5.0 mg/minute, between about 0.5 mL minute and about 14 mL/minute,between about 1.0 mL/minute and about 25.0 mL/minute, or between about1.0 mL/minute and about 15.0 mL/minute). The volume of elution bufferused to elute the recombinant protein from each of the at least onechromatographic column or chromatographic membrane that can be used toperform the unit operation of purifying the recombinant protein can be,e.g., between about 1× column volume (CV) to about 25×CV (e.g., betweenabout 1×CV to about 20×CV, between about 15×CV and about 25×CV, betweenabout 1×CV to about 14×CV, about 1×CV to about 13×CV, about 1×CV toabout 12×CV, about 1× CV to about 11×CV, about 2×CV to about 11×CV,about 3×CV to about 11×CV, about 4×CV to about 11×CV, about 5×CV toabout 11×CV, or about 5×CV to about 10×CV). The total time of theeluting can be, e.g., between about 2 minutes to about 3 hours (e.g.,between about 2 minutes to about 2.5 hours, between about 2 minutes toabout 2.0 hours, between about 2 minutes to about 1.5 hours, betweenabout 2 minutes to about 1.5 hours, between about 2 minutes to about1.25 hours, between about 2 minutes to about 1.25 hours, between about 2minutes to about 1 hour, between about 2 minutes and about 40 minutes,between about 10 minutes and about 40 minutes, between about 20 minutesand about 40 minutes, or between about 30 minutes and 1.0 hour).Non-limiting examples of elution buffers that can be used in thesemethods will depend on the resin and/or the biophysical properties ofthe recombinant protein. For example, an elution buffer can include adifferent concentration of salt (e.g., increased salt concentration), adifferent pH (e.g., an increased or decreased salt concentration), or amolecule that will compete with the recombinant protein for binding tothe resin. Examples of such elution buffers for each of the exemplarycapture mechanisms described herein are well known in the art.

Following the elution of the recombinant protein from the at least onechromatographic column or chromatographic membrane used to perform theunit operation of purifying the recombinant protein, and before the nextvolume of fluid including a recombinant protein can be loaded onto theat least one chromatographic column or chromatographic membrane, the atleast one chromatography column or chromatographic membrane must beequilibrated using an regeneration buffer. The regeneration buffer canbe passed through the at least one chromatography column orchromatographic membrane used to perform the unit operation of purifyingthe recombinant protein at a flow rate of, e.g., between about 0.2mL/minute to about 25 mL/minute (e.g., between about 0.2 mL/minute toabout 20 mL/minute, between about 0.5 mL/minute to about 20 mL/minute,between about 0.2 mL/minute to about 15 mL/minute, between about 0.5mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL/minute and about 6.0 mL/minute, betweenabout 1.0 mL/minute and about 5.0 mg/minute, between about 0.5 mL minuteand about 14 mL/minute, between about 1.0 mL/minute and about 25.0mL/minute, between about 5.0 mL/minute to about 15.0 mL/minute, orbetween about 1.0 mL/minute and about 15.0 mL/minute). The volume ofregeneration buffer used to equilibrate the at least one chromatographycolumn or chromatographic membrane that includes a resin that can beused to perform the unit operation of purifying the recombinant proteincan be, e.g., between about 1× column volume (CV) to about 15×CV (e.g.,between about 1×CV to about 14×CV, between about 1×CV to about 13×CV,between about 1×CV to about 12×CV, between about 1×CV to about 11×CV,between about 2×CV to about 11×CV, between about 3×CV to about 11×CV,between about 2×CV to about 5×CV, between about 2.5×CV to about 7.5×CV,between about 4×CV to about 11×CV, between about 5×CV to about 11×CV, orbetween about 5×CV to about 10×CV). The concentration of recombinantprotein in the eluate of the at least one chromatography column orchromatographic membrane used to perform the unit operation of purifyingthe recombinant protein can be, e.g., between about 0.05 mg/mL to about100 mg/mL recombinant protein (e.g., between about 0.1 mg/mL to about 90mg/mL, between about 0.1 mg/mL to about 80 mg/mL, between about 0.1mg/mL to about 70 mg/mL, between about 0.1 mg/mL to about 60 mg/mL,between about 0.1 mg/mL to about 50 mg/mL, between about 0.1 mg/mL toabout 40 mg/mL, between about 2.5 mg/mL and about 7.5 mg/mL, betweenabout 0.1 mg/mL to about 30 mg/mL, between about 0.1 mg/mL to about 20mg/mL, between 0.5 mg/mL to about 20 mg/mL, between about 0.1 mg/mL toabout 15 mg/mL, between about 0.5 mg/mL to about 15 mg/mL, between about0.1 mg/mL to about 10 mg/mL, or between about 0.5 mg/mL to about 10mg/mL recombinant protein).

The at least one chromatography column or chromatographic membrane thatcan be used to perform the unit operation of polishing the recombinantprotein can include a resin that can be used to perform cation exchange,anion exchange, or molecular sieve chromatography. As can be appreciatedin the art, polishing a recombinant protein using the at least onechromatography column or chromatography membrane that can be used toperform the unit operation of polishing the recombinant protein caninclude, e.g., the steps of loading, chasing, and regenerating the atleast one chromatography column or chromatographic membrane that can beused to perform the unit operation of polishing the recombinant protein.For example, when the steps of loading, chasing, and regenerating areused to perform the polishing, the recombinant protein does not bind theresin in the at least one chromatography column or chromatographymembrane that is used to perform the unit operation of polishing therecombinant protein, and the recombinant protein is eluted from the atleast one chromatography column or chromatographic membrane in theloading and chasing steps, and the regenerating step is used to removeany impurities from the at least one chromatography column orchromatographic membrane before additional fluid including therecombinant protein can be loaded onto the at least one chromatographycolumn or chromatographic membrane. Exemplary flow rates and buffervolumes to be used in each of the loading, chasing, and regeneratingsteps are described below.

The size, shape, and volume of the at least one chromatography column orchromatography membrane that can be used to perform the unit operationof polishing the recombinant protein, and/or the size and shape of theat least one chromatographic membrane that can be used to perform theunit operation of polishing the recombinant protein can any ofcombination of the exemplary sizes, shapes, and volumes ofchromatography columns or chromatographic membranes described herein.For example, the size of the at least one chromatography column orchromatographic membrane that can be used to perform the unit operationof polishing the recombinant protein can have a volume of, e.g., betweenabout 0.5 mL to about 200 mL (e.g., between about 0.5 mL to about 180mL, between about 0.5 mL to about 160 mL, between about 0.5 mL to about140 mL, between about 0.5 mL to about 120 mL, between about 0.5 mL toabout 100 mL, between about 0.5 mL to about 80 mL, between about 0.5 mLto about 60 mL, between about 0.5 mL to about 40 mL, between about 5.0mL to about 40 mL, between about 0.5 mL to about 30 mL, between about5.0 mL to about 30 mL, between about 0.5 mL to about 25 mL, betweenabout 0.2 mL to about 10 mL, or between about 0.2 mL to about 5 mL). Theflow rate of the fluid including the recombinant protein as it is loadedonto the at least one chromatography column or chromatographic membranethat can be used to perform the unit operation of polishing therecombinant protein can be, e.g., between about 0.1 mL/minute to about25 mL/minute (e.g., between about 0.1 mL/minute to about 12.5 mL/minute,between about 0.1 mL/minute to about 10.0 mL/minute, between about 0.1mL/minute to about 8.0 mL/minute, between about 0.1 mL/minute to about 6mL/minute, between about 0.1 mL/minute to 4 mL/minute, between about 0.1mL/minute to about 3 mL/minute, between about 2 mL/minute and about 6mL/minute, between about 0.1 mL/minute to about 2 mL/minute, or about0.2 mL/minute to about 4 mL/minute). The total volume of fluid includinga recombinant protein loaded onto the at least one chromatography columnor chromatographic membrane that can be used to perform the unitoperation of polishing the recombinant protein can be, e.g., betweenabout 1.0 mL to about 250 mL (e.g., between about 1.0 mL to about 225mL, between about 1.0 mL to about 200 mL, between about 1.0 mL to about175 mL, between about 1.0 mL to about 150 mL, between about 100 mL toabout 125 mL, between about 100 mL to about 150 mL, between about 1.0 mLto about 150 mL, between about 1.0 mL to about 125 mL, between about 1.0mL to about 100 mL, between about 1.0 mL to about 75 mL, between about1.0 mL to about 50 mL, or between about 1.0 mL to about 25 mL). Theresin in the at least one chromatography column or chromatographicmembrane used to perform the polishing can be an anion exchange orcation exchange resin. The resin in the at least one chromatographycolumn or chromatographic membrane that is used to perform the unitoperation of polishing can be a cationic exchange resin (e.g.,Sartobind® Q resin, Sartorius, Goettingen, Germany).

Following the loading step, a chasing step is performed (e.g., a chasebuffer is passed through the at least one chromatography membrane orchromatographic membrane to collect the recombinant protein which doesnot substantially bind to the at least one chromatography column orchromatographic membrane). In these examples, the chase buffer can bepassed through the at least one chromatography column or chromatographicmembrane at a flow rate of between about 0.2 mL/minute to about 50mL/minute (e.g., between about 1 mL/minute to about 40 mL/minute,between about 1 mL/minute to about 30 mL/minute, between about 5mL/minute to about 45 mL/minute, between about 10 mL/minute to about 40mL/minute, between about 0.2 mL/minute to about 20 mL/minute, betweenabout 0.5 mL/minute to about 20 mL/minute, between about 0.2 mL/minuteto about 15 mL/minute, between about 0.5 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 10 mL/minute, betweenabout 0.5 mL minute and about 14 mL/minute, between about 1.0 mL/minuteand about 25.0 mL/minute, or between about 1.0 mL/minute and about 15.0mL/minute). The volume of chase buffer used can be, e.g., between about1× column volume (CV) to about 100×CV (e.g., between about 1×CV to about90×CV, between about 1×CV to about 80×CV, between about 1×CV to about70×CV, between about 1×CV to about 60×CV, between about 1× to about50×CV, between about 1×CV to about 40×CV, between about 1×CV to about30×CV, between about 1×CV to about 20×CV, between about 1×CV to about15×CV, between about 5×CV to about 20×CV, between about 5×CV to about30×CV, between about 1×CV to about 14×CV, about 1×CV to about 13×CV,about 1×CV to about 12×CV, about 1×CV to about 11×CV, about 2×CV toabout 11×CV, about 3×CV to about 11×CV, about 4×CV to about 11×CV, about2.5×CV to about 5.0×CV, about 5×CV to about 11×CV, or about 5×CV toabout 10×CV). The total time of the chasing can be, e.g., between about1 minute to about 3 hours (e.g., between about 1 minute to about 2.5hours, between about 1 minute to about 2.0 hours, between about 1 minuteto about 1.5 hours, between about 2 minutes to about 1.5 hours, betweenabout 1 minute to about 1.25 hours, between about 2 minutes to about1.25 hours, between about 1 minute to about 5 minutes, between about 1minute to about 10 minutes, between about 2 minutes to about 4 minutes,between about 30 minutes to about 1 hour, between about 2 minutes toabout 10 minutes, between about 2 minutes to about 15 minutes, orbetween about 2 minutes to about 30 minutes). The combined concentrationof recombinant protein present in the eluate coming through the columnin the loading step and the chasing step can be, e.g., between about 0.1mg/mL to about 100 mg/mL recombinant protein (e.g., between about 0.1mg/mL to about 90 mg/mL, between about 0.1 mg/mL to about 80 mg/mL,between about 0.1 mg/mL to about 70 mg/mL, between about 0.1 mg/mL toabout 60 mg/mL, between about 0.1 mg/mL to about 50 mg/mL, between about0.1 mg/mL to about 40 mg/mL, between about 2.5 mg/mL and about 7.5mg/mL, between about 0.1 mg/mL to about 30 mg/mL, between about 0.1mg/mL to about 20 mg/mL, between 0.5 mg/mL to about 20 mg/mL, betweenabout 0.1 mg/mL to about 15 mg/mL, between about 0.5 mg/mL to about 15mg/mL, between about 0.1 mg/mL to about 10 mg/mL, between about 0.5mg/mL to about 10 mg/mL, or between about 1 mg/mL and about 5 mg/mLrecombinant protein).

Following the chasing step and before the next volume of fluid includingthe recombinant protein can be loaded onto the at least onechromatographic column or chromatographic membrane that can be used toperform the unit operation of polishing, the at least one chromatographycolumn or chromatographic membrane must be regenerated using aregeneration buffer. The regeneration buffer can be passed through theat least one chromatography column or chromatographic membrane that canbe used to perform the unit operation of polishing the recombinantprotein at a flow rate of, e.g., between about 0.2 mL/minute to about 50mL/minute (e.g., between about 1 mL/minute to about 40 mL/minute,between about 1 mL/minute to about 30 mL/minute, between about 5mL/minute to about 45 mL/minute, between about 10 mL/minute to about 40mL/minute, between about 0.2 mL/minute to about 20 mL/minute, betweenabout 0.5 mL/minute to about 20 mL/minute, between about 0.2 mL/minuteto about 15 mL/minute, between about 0.5 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 10 mL/minute, betweenabout 0.5 mL minute and about 14 mL/minute, between about 1.0 mL/minuteand about 25.0 mL/minute, or between about 1.0 mL/minute and about 15.0mL/minute). The volume of regeneration buffer used to regenerate the atleast one chromatography column or chromatographic membrane that can beused to perform the unit operation of polishing can be, e.g., betweenabout 1× column volume (CV) to about 500×CV (e.g., between about 1×CV toabout 450×CV, between about 1×CV to about 400×CV, between about 1×CV toabout 350×CV, between about 1×CV to about 300×CV, between about 1×CV toabout 250×CV, between about 1×CV to about 200×CV, between about 1×CV toabout 150×CV, between about 1×CV to about 100×CV, between about 1×CV toabout 90×CV, between about 1×CV to about 80×CV, between about 1×CV toabout 70×CV, between about 1×CV to about 60×CV, between about 1× toabout 50×CV, between about 1×CV to about 40×CV, between about 1×CV toabout 30×CV, between about 1×CV to about 20×CV, between about 1×CV toabout 15×CV, between about 5×CV to about 20×CV, between about 5×CV toabout 30×CV, between about 1×CV to about 14×CV, about 1×CV to about13×CV, about 1×CV to about 12×CV, about 1×CV to about 11×CV, about 2×CVto about 11×CV, about 3×CV to about 11×CV, about 4×CV to about 11×CV,about 2.5×CV to about 5.0×CV, about 5×CV to about 11×CV, or about 5×CVto about 10×CV).

In other examples, the one or more chromatography column(s) and/orchromatographic membranes used to perform the unit operation ofpolishing include a resin that selectively binds or retains theimpurities present in a fluid including the recombinant protein, andinstead of regenerating the one or more column(s) and/or membrane(s),the one or more column(s) and/or membrane(s) are replaced (e.g.,replaced with a substantially similar column(s) and/or membrane(s)) oncethe binding capacity of the resin in the one or more column(s) and/ormembrane(s) has been reached or is substantially close to being reached.

In some examples of these processes described herein, the MCCS2 includesa PCCS including three chromatography columns and one chromatographicmembrane, e.g., where the three chromatography columns in the PCCSperform the unit operation of purifying the recombinant protein (e.g.,using at least one chromatography column(s) that can be used to performthe unit of operation of purifying the protein) and the chromatographicmembrane in the PCCS performs the unit operation of polishing therecombinant protein. In these examples, the chromatographic membrane inthe PCCS that can be used to perform the unit operation of polishing thetherapeutic protein can be any of the exemplary chromatographicmembranes described herein that can be used to perform the unitoperation of polishing the recombinant protein. Any of the columnswitching methods described herein can be used to determine when thefirst three chromatography columns and the chromatographic membrane inthe PCCS in this example can be switched.

Some embodiments of this example can further include a step of adjustingthe ionic concentration and/or pH of the eluate from the threechromatographic columns in the PCCS before the eluate is fed into thechromatographic membrane in the PCCS. As described herein, the ionicconcentration and/or pH of the eluate from the three chromatographycolumns in PCCS can be adjusted (before it is fed into thechromatographic membrane in the PCCS in this example)) by adding abuffer to the eluate of the three chromatography columns in the PCCS(e.g., through the use of an in-line buffer adjustment reservoir). Thebuffer can be added to the eluate at a flow rate of, e.g., between about0.1 mL/minute to about 15 mL/minute (e.g., between about 0.1 mL/minuteto about 12.5 mL/minute, between about 0.1 mL/minute to about 10.0mL/minute, between about 0.1 mL/minute to about 8.0 mL/minute, betweenabout 0.1 mL/minute to about 6 mL/minute, between about 0.1 mL/minute to4 mL/minute, or between about 0.5 mL/minute to about 5 mL/minute).

These examples can further include a step of holding or storing theeluate from the three chromatography columns in the PCCS in this exampleprior to feeding the eluate into the chromatographic membrane(chromatographic membrane that can be used to perform the unit operationof polishing the recombinant protein). As described herein, this holdingor storing step can be performed using any of the reservoirs (e.g.,back-up tanks) described herein.

These examples can also include a step of filtering the eluate from thechromatographic membrane in the exemplary PCCS system (eluate of thechromatographic membrane that can be used to perform the unit operationof polishing the recombinant protein). Any of the exemplary filters ormethods for filtration described herein can be used to filter the eluatefrom the chromatographic membrane in this exemplary PCCS (eluate of thechromatographic membrane that can be used to perform the unit operationof polishing the recombinant protein).

As can be appreciated by those in the art, the purified recombinantprotein can be periodically eluted from the MCCS or MCCS2 using any ofthe processes described herein. For example, any of the processesdescribed herein can elute the purified recombinant protein for aduration of, e.g., between about 30 seconds and about 5 hours (e.g.,between about 1 minute and about 4 hours, between about 1 minute andabout 3 hours, between about 1 minute and about 2 hours, between about 1minute or about 1.5 hours, between about 1 minute and about 1 hour, orbetween about 1 minute and about 30 minutes) at a frequency of, e.g.,between about 1 minute and about 6 hours (e.g., between about 1 minuteand about 5 hours, between about 1 minute and about 4 hours, betweenabout 1 minute and about 3 hours, between about 1 minute and 2 hours,between about 1 minute and 1 hour, or between about 1 minute and 30minutes), depending on, e.g., the chromatography column(s) and/orchromatographic membrane(s) used in the MCCS or the MCCS1 and MCCS2.

Culturing Methods

Some of the processes described herein further include a step ofculturing cells (e.g., recombinant mammalian cells) that secrete arecombinant protein in a bioreactor (e.g., a perfusion or fed-batchbioreactor) that includes a liquid culture medium, wherein a volume ofthe liquid culture medium that is substantially free of cells (e.g.,mammalian cells) is continuously or periodically removed from thebioreactor (e.g., perfusion bioreactor) and fed into the MCCS or MCCS1.The bioreactor can have a volume of, e.g., between about 1 L to about10,000 L (e.g., between about 1 L to about 50 L, between about 50 L toabout 500 L, between about 500 L to about 1000 L, between 500 L to about5000 L, between about 500 L to about 10,000 L, between about 5000 L toabout 10,000 L, between about 1 L and about 10,000 L, between about 1 Land about 8,000 L, between about 1 L and about 6,000 L, between about 1L and about 5,000 L, between about 100 L and about 5,000 L, betweenabout 10 L and about 100 L, between about 10 L and about 4,000 L,between about 10 L and about 3,000 L, between about 10 L and about 2,000L, or between about 10 L and about 1,000 L). The amount of liquidculture medium present in a bioreactor can be, e.g., between aboutbetween about 0.5 L to about 5,000 L (e.g., between about 0.5 L to about25 L, between about 25 L to about 250 L, between about 250 L to about500 L, between 250 L to about 2500 L, between about 250 L to about 5,000L, between about 2500 L to about 5,000 L, between about 0.5 L and about5,000 L, between about 0.5 L and about 4,000 L, between about 0.5 L andabout 3,000 L, between about 0.5 L and about 2,500 L, between about 50 Land about 2,500 L, between about 5 L and about 50 L, between about 5 Land about 2,000 L, between about 5 L and about 1,500 L, between about 5L and about 1,000 L, or between about 5 L and about 500 L). Culturingcells can be performed, e.g., using a fed-batch bioreactor or aperfusion bioreactor. Non-limiting examples and different aspects ofculturing cells (e.g., culturing mammalian cells) are described belowand can be used in any combination.

Cells

The cells that are cultured in some of the processes described hereincan be bacteria (e.g., gram negative bacteria), yeast (e.g.,Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha,Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica, orArxula adeninivorans), or mammalian cells. The mammalian cell can be acell that grows in suspension or an adherent cell. Non-limiting examplesof mammalian cells that can be cultured in any of the processesdescribed herein include: Chinese hamster ovary (CHO) cells (e.g., CHODG44 cells or CHO-K1s cells), Sp2.0, myeloma cells (e.g., NS/0),B-cells, hybridoma cells, T-cells, human embryonic kidney (HEK) cells(e.g., HEK 293E and HEK 293F), African green monkey kidney epithelialcells (Vero) cells, and Madin-Darby Canine (Cocker Spaniel) kidneyepithelial cells (MDCK) cells. In some examples where an adherent cellis cultured, the culture can also include a plurality of microcarriers(e.g., microcarriers that include one or more pores). Additionalmammalian cells that can be cultured in any of the processes describedherein are known in the art.

The mammalian cell can include a recombinant nucleic acid (e.g., anucleic acid stably integrated in the mammalian cell's genome) thatencodes a recombinant protein (e.g., a recombinant protein).Non-limiting examples of recombinant nucleic acids that encode exemplaryrecombinant proteins are described below, as are recombinant proteinsthat can be produced using the methods described herein. In someinstances, the mammalian cell that is cultured in a bioreactor (e.g.,any of the bioreactors described herein) was derived from a largerculture.

A nucleic acid encoding a recombinant protein can be introduced into amammalian cell using a wide variety of methods known in molecularbiology and molecular genetics. Non-limiting examples includetransfection (e.g., lipofection), transduction (e.g., lentivirus,adenovirus, or retrovirus infection), and electroporation. In someinstances, the nucleic acid that encodes a recombinant protein is notstably integrated into a chromosome of the mammalian cell (transienttransfection), while in others the nucleic acid is integrated.Alternatively or in addition, the nucleic acid encoding a recombinantprotein can be present in a plasmid and/or in a mammalian artificialchromosome (e.g., a human artificial chromosome). Alternatively or inaddition, the nucleic acid can be introduced into the cell using a viralvector (e.g., a lentivirus, retrovirus, or adenovirus vector). Thenucleic acid can be operably linked to a promoter sequence (e.g., astrong promoter, such as a (β-actin promoter and CMV promoter, or aninducible promoter). A vector including the nucleic acid can, ifdesired, also include a selectable marker (e.g., a gene that confershygromycin, puromycin, or neomycin resistance to the mammalian cell).

In some instances, the recombinant protein is a secreted protein and isreleased by the mammalian cell into the extracellular medium (e.g., thefirst and/or second liquid culture medium). For example, a nucleic acidsequence encoding a soluble recombinant protein can include a sequencethat encodes a secretion signal peptide at the N- or C-terminus of therecombinant protein, which is cleaved by an enzyme present in themammalian cell, and subsequently released into the extracellular medium(e.g., the first and/or second liquid culture medium).

Culture Media

Liquid culture media are known in the art. The liquid culture media(e.g., a first and/or second tissue culture medium) can be supplementedwith a mammalian serum (e.g., fetal calf serum and bovine serum), and/ora growth hormone or growth factor (e.g., insulin, transferrin, andepidermal growth factor). Alternatively or in addition, the liquidculture media (e.g., a first and/or second liquid culture medium) can bea chemically-defined liquid culture medium, an animal-derived componentfree liquid culture medium, a serum-free liquid culture medium, or aserum-containing liquid culture medium. Non-limiting examples ofchemically-defined liquid culture media, animal-derived component freeliquid culture media, serum-free liquid culture media, andserum-containing liquid culture media are commercially available.

A liquid culture medium typically includes an energy source (e.g., acarbohydrate, such as glucose), essential amino acids (e.g., the basicset of twenty amino acids plus cysteine), vitamins and/or other organiccompounds required at low concentrations, free fatty acids, and/or traceelements. The liquid culture media (e.g., a first and/or second liquidculture medium) can, if desired, be supplemented with, e.g., a mammalianhormone or growth factor (e.g., insulin, transferrin, or epidermalgrowth factor), salts and buffers (e.g., calcium, magnesium, andphosphate salts), nucleosides and bases (e.g., adenosine, thymidine, andhypoxanthine), protein and tissue hydrolysates, and/or any combinationof these additives.

A wide variety of different liquid culture media that can be used toculture cells (e.g., mammalian cells) in any of the methods describedherein are known in the art. Medium components that also may be usefulin the present processes include, but are not limited to,chemically-defined (CD) hydrolysates, e.g., CD peptone, CD polypeptides(two or more amino acids), and CD growth factors. Additional examples ofliquid tissue culture medium and medium components are known in the art.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium described herein can be thesame type of media or different media.

Additional Features of Exemplary Bioreactors

The interior surface of any of the bioreactors described herein may haveat least one coating (e.g., at least one coating of gelatin, collagen,poly-L-ornithine, polystyrene, and laminin), and as is known in the art,one or more ports for the sparging of O₂, CO₂, and N₂ into the liquidculture medium, and a stir mechanism for agitating the liquid culturemedium. The bioreactor can incubate the cell culture in a controlledhumidified atmosphere (e.g., at a humidity of greater than 20%, 30%,40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or a humidity of 100%).The bioreactor can also be equipped with a mechanical device that iscapable of removing a volume of liquid culture medium from thebioreactor and optionally, a filter within the mechanical device thatremoves the cells from the liquid culture medium during the process oftransfer of the liquid culture medium out of the bioreactor (e.g., anATF system or the cell filtering system described in U.S. ProvisionalPatent Application Ser. No. 61/878,502).

Temperature

The step of culturing of mammalian cells can be performed at atemperature of about 31° C. to about 40° C. Skilled practitioners willappreciate that the temperature can be changed at specific time point(s)in during the culturing step, e.g., on an hourly or daily basis. Forexample, the temperature can be changed or shifted (e.g., increased ordecreased) at about one day, two days, three days, four days, five days,six days, seven days, eight days, nine days, ten days, eleven days,twelve days, fourteen days, fifteen days, sixteen days, seventeen days,eighteen days, nineteen days, or about twenty days or more after theinitial seeding of the bioreactor with the cell (e.g., mammalian cell).For example, the temperature can be shifted upwards (e.g., a change ofup to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to or about 20°C.). For example, the temperature can be shifted downwards (e.g., achange of up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to orabout 20° C.).

CO₂

The culturing step described herein can further include exposing theliquid culture medium in the bioreactor to an atmosphere including atmost or about 15% CO₂ (e.g., at most or about 14% CO₂, 12% CO₂, 10% CO₂,8% CO₂, 6% CO₂, 5% CO₂, 4% CO₂, 3% CO₂, 2% CO₂, or at most or about 1%CO₂).

Perfusion Bioreactor

The culturing step described herein can be performed using a perfusionbioreactor. Culturing a cell (e.g., a mammalian cell) in a perfusionbioreactor includes the removal from the bioreactor of a first volume ofa first liquid culture medium (e.g., including any concentration ofmammalian cells, e.g., a first volume of a first liquid culture mediumthat is substantially free of cells), and adding to the first liquidculture medium a second volume of a second liquid culture medium.Removal and adding can be performed simultaneously or sequentially, or acombination of the two. Further, removal and adding can be performedcontinuously (e.g., at a rate that removes and replaces a volume ofbetween 0.1% to 800% (e.g., between 1% and 700%, between 1% and 600%,between 1% and 500%, between 1% and 400%, between 1% and 350%, between1% and 300%, between 1% and 250%, between 1% and 100%, between 100% and200%, between 5% and 150%, between 10% and 50%, between 15% and 40%,between 8% and 80%, or between 4% and 30%) of the volume of thebioreactor or the first liquid culture medium volume over any given timeperiod (e.g., over a 24-hour period, over an incremental time period ofabout 1 hour to about 24 hours, or over an incremental time period ofgreater than 24 hours)) or periodically (e.g., once every third day,once every other day, once a day, twice a day, three times a day, fourtimes a day, or five times a day), or any combination thereof. Whereperformed periodically, the volume that is removed or replaced (e.g.,within about a 24-hour period, within an incremental time period ofabout 1 hour to about 24 hours, or within an incremental time period ofgreater than 24 hours) can be, e.g., between 0.1% to 800% (e.g., between1% and 700%, between 1% and 600%, between 1% and 500%, between 1% and400%, between 1% and 300%, between 1% and 200%, between 1% and 100%,between 100% and 200%, between 5% and 150%, between 10% and 50%, between15% and 40%, between 8% and 80%, or between 4% and 30%) of the volume ofthe bioreactor or the first liquid culture medium volume. The firstvolume of the first liquid culture medium removed and the second volumeof the second liquid culture medium added can in some instances be heldapproximately the same over each 24-hour period (or, alternatively, anincremental time period of about 1 hour to about 24 hours or anincremental time period of greater than 24 hours) over the entire orpart of the culturing period. As is known in the art, the rate at whichthe first volume of the first liquid culture medium is removed(volume/unit of time) and the rate at which the second volume of thesecond liquid culture medium is added (volume/unit of time) can bevaried. The rate at which the first volume of the first liquid culturemedium is removed (volume/unit of time) and the rate at which the secondvolume of the second liquid culture medium is added (volume/unit oftime) can be about the same or can be different.

Alternatively, the volume removed and added can change (e.g., graduallyincrease) over each 24-hour period (or alternatively, an incrementaltime period of between 1 hour and about 24 hours or an incremental timeperiod of greater than 24 hours) during the culturing period. Forexample the volume of the first liquid culture medium removed and thevolume of the second liquid culture medium added within each 24-hourperiod (or alternatively, an incremental time period of between about 1hour and above 24 hours or an incremental time period of greater than 24hours) over the culturing period can be increased (e.g., gradually orthrough staggered increments) over the culturing period from a volumethat is between 0.5% to about 20% of the bioreactor volume or the firstliquid culture medium volume to about 25% to about 150% of thebioreactor volume or the first liquid culture medium volume.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium can be the same type ofmedia. In other instances, the first liquid culture medium and thesecond liquid culture medium can be different.

The first volume of the first liquid culture medium can be removed,e.g., by a mechanical system that can remove the first volume of thefirst liquid culture medium from the bioreactor (e.g., the first volumeof the first liquid culture medium that is substantially free of cellsfrom the bioreactor). Alternatively or in addition, the first volume ofthe first liquid culture medium can be removed by seeping or gravityflow of the first volume of the first liquid culture medium through asterile membrane with a molecular weight cut-off that excludes the cell(e.g., mammalian cell).

The second volume of the second liquid culture medium can be added tothe first liquid culture medium in an automated fashion, e.g., byperfusion pump.

In some instances, removing the first volume of the first liquid culturemedium (e.g., a first volume of the first liquid culture medium that issubstantially free of mammalian cells) and adding to the first liquidculture medium a second volume of the second liquid culture medium doesnot occur within at least 1 hour (e.g., within 2 hours, within 3 hours,within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8hours, within 9 hours, within 10 hours, within 12 hours, within 14hours, within 16 hours, within 18 hours, within 24 hours, within 36hours, within 48 hours, within 72 hours, within 96 hours, or after 96hours) of the seeding of the bioreactor with a mammalian cell.

Fed-Batch Bioreactor

The culturing step described herein can be performed using a fed-batchbioreactor. Culturing a cell in a fed-batch bioreactor includes, overthe majority of the culturing period, the addition (e.g., periodic orcontinuous addition) to the first liquid culture medium of a secondvolume of a second liquid culture medium. The adding of the secondliquid culture medium can be performed continuously (e.g., at a ratethat adds a volume of between 0.1% to 300% (e.g., between 1% and 250%,between 1% and 100%, between 100% and 200%, between 5% and 150%, between10% and 50%, between 15% and 40%, between 8% and 80%, or between 4% and30%) of the volume of the bioreactor or the first liquid culture mediumvolume over any given time period (e.g., over a 24-hour period, over anincremental time period of about 1 hour to about 24 hours, or over anincremental time period of greater than 24 hours)) or periodically(e.g., once every third day, once every other day, once a day, twice aday, three times a day, four times a day, or five times a day), or anycombination thereof. Where performed periodically, the volume that isadded (e.g., within about a 24-hour period, within an incremental timeperiod of about 1 hour to about 24 hours, or within an incremental timeperiod of greater than 24 hours) can be, e.g., between 0.1% to 300%(e.g., between 1% and 200%, between 1% and 100%, between 100% and 200%,between 5% and 150%, between 10% and 50%, between 15% and 40%, between8% and 80%, or between 4% and 30%) of the volume of the bioreactor orthe first liquid culture medium volume. The second volume of the secondliquid culture medium added can in some instances be held approximatelythe same over each 24-hour period (or, alternatively, an incrementaltime period of about 1 hour to about 24 hours or an incremental timeperiod of greater than 24 hours) over the entire or part of theculturing period. As is known in the art, the rate at which the secondvolume of the second liquid culture medium is added (volume/unit oftime) can be varied over the entire or part of the culturing period. Forexample, the volume of the second liquid culture medium added can change(e.g., gradually increase) over each 24-hour period (or alternatively,an incremental time period of between 1 hour and about 24 hours or anincremental time period of greater than 24 hours) during the culturingperiod. For example the volume of the second liquid culture medium addedwithin each 24-hour period (or alternatively, an incremental time periodof between about 1 hour and above 24 hours or an incremental time periodof greater than 24 hours) over the culturing period can be increased(e.g., gradually or through staggered increments) over the culturingperiod from a volume that is between 0.5% to about 20% of the bioreactorvolume or the first liquid culture medium volume to about 25% to about150% of the bioreactor volume or the first liquid culture medium volume.The rate at which the second volume of the second liquid culture mediumis added (volume/unit of time) can be about the same over the entire orpart of the culturing period.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium can be the same type ofmedia. In other instances, the first liquid culture medium and thesecond liquid culture medium can be different. The volume of the secondliquid culture medium can be added to the first liquid culture medium inan automated fashion, e.g., by perfusion pump.

In some instances, adding to the first liquid culture medium a secondvolume of the second liquid culture medium does not occur within atleast 1 hour (e.g., within 2 hours, within 3 hours, within 4 hours,within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9hours, within 10 hours, within 12 hours, within 14 hours, within 16hours, within 18 hours, within 24 hours, within 36 hours, within 48hours, within 72 hours, within 96 hours, or after 96 hours) of theseeding of the bioreactor with a mammalian cell. The cell culture mediumin fed-batch cultures is typically harvested at the end of cultureperiod and used in any of the processes described herein, however, thecell culture medium in fed-batch cultures can also be harvested at oneor more time points during the culturing period and used in any of theprocesses described herein.

Skilled practitioners will appreciate that any of the various cultureparameters (e.g., containers, volumes, rates or frequencies of replacingculture volumes, agitation frequencies, temperatures, media, and CO₂concentrations) can be used in any combination in to perform thesemethods. Further, any of the mammalian cells described herein or knownin the art can be used to produce a recombinant protein.

Exemplary Biological Manufacturing Systems

Examples of biological manufacturing systems useful for performing theprocesses described herein and that include a MCCS or a MCCS1 and MCCS2are described in U.S. Provisional Patent Application Ser. Nos.61/775,060 and 61/856,390 (incorporated by reference). In theseexemplary systems, at least one (e.g., at least two, three, four, five,or six) at least one reduced bioburden packed chromatography columnprovided herein is present in the MCCS or in the MCCS1 and/or MCCS2. Forexample, the entire system can include a total of two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of thereduced bioburden packed chromatography columns provided herein. Forexample, the MCCS, MCCS1, and/or MCCS2 can include (or can each include)one, two, three, four, five, six, seven, eight, nine, or ten of thereduced bioburden packed chromatography columns provided herein.

For example, useful systems can include a MCCS1 that includes an inletand a MCCS2 that includes an outlet, or an MCCS that includes an inletand an outlet. In some embodiments, the MCCS1 and MCCS2 are in fluidcommunication with each other. These systems can also be configured suchthat fluid can be passed into the inlet, through the MCCS1 and MCCS2,and exit the manufacturing system through the outlet. These systemsprovide for the continuous and time-efficient production of atherapeutic drug substance from a liquid culture medium. For example,the elapsed time between feeding a fluid (e.g., a liquid culture medium)including a therapeutic protein into the MCCS1 and eluting purifiedrecombinant protein (e.g., therapeutic protein drug substance) from theoutlet of the MCCS2 can be, e.g., between about 4 hours and about 48hours, inclusive.

Some exemplary systems do not include a break tank. In others, thesystem can include a maximum of 1, 2, 3, 4, or 5 break tank(s) in theentire system (e.g., where each break tank only holds a therapeuticprotein for a total time period of, e.g., between about 5 minutes andabout 6 hours, inclusive). The break tank(s) can have a capacity that isbetween 1 mL and about 300 mL, inclusive. Any break tank(s) disposed inthe system such that fluid enters the break tank(s) prior to enteringMCCS1 or MCCS can have a capacity that is between 1 mL and about 100%,inclusive, of the loading volume of the first column of the MCCS1 orMCCS, respectively. Any break tanks(s) disposed in the system such thatfluid enters the break tank(s) prior to entering the MCCS2 (and afterexiting the MCCS1) can have a capacity that is, e.g., between 1 mL andabout 100%, inclusive, of the loading volume of the first column of theMCCS2.

Additional Exemplary System Structures and Features

The MCCS or MCCS1 can include an inlet through which fluid (e.g., aliquid culture medium that is substantially free of cells) can be passedinto the MCCS or MCCS1, respectively. The inlet can be any structureknown in the art for such purposes. It can include, e.g., a threading,ribbing, or a seal that allows for a fluid conduit to be inserted, suchthat after insertion of the fluid conduit into the inlet, fluid willenter the MCCS or MCCS1 through the inlet without significant seepage offluid out of the inlet. Non-limiting inlets that can be used in thepresent systems are known and would be understood by those in the art.

The MCCS or MCCS1 can include at least two chromatography columns, atleast two chromatographic membranes, or at least one chromatographycolumn and at least one chromatographic membrane, and an inlet. The MCCSor MCCS1 can be any of the exemplary MCCSs described herein, or have oneor more of any of the exemplary features of an MCCS (in any combination)described herein. The chromatography column(s) and/or thechromatographic membrane(s) present in the MCCS or MCCS1 can have one ormore of any of the exemplary shapes, sizes, volumes (bed volumes),and/or unit operation(s) described herein.

The chromatography column(s) and/or the chromatographic membrane(s)present in the MCCS or MCCS1 can include one or more of any of theexemplary resins described herein or known in the art. For example, theresin included in one or more of the chromatography column(s) and/orchromatographic membrane(s) present in the MCCS or MCCS1 can be a resinthat utilizes a capture mechanism (e.g., protein A-binding capturemechanism, protein G-binding capture mechanism, antibody- or antibodyfragment-binding capture mechanism, substrate-binding capture mechanism,cofactor-binding capture mechanism, an aptamer-binding capturemechanism, and/or a tag-binding capture mechanism). The resin includedin one or more of the chromatography column(s) and/or chromatographicmembrane(s) of the MCCS or MCCS1 can be a cation exchange resin, ananion exchange resin, a molecular sieve resin, or a hydrophobicinteraction resin, or any combination thereof. Additional examples ofresins that can be used to purify a recombinant protein are known in theart, and can be included in one or more of the chromatography column(s)and/or chromatographic membrane(s) present in the MCCS or MCCS1. Thechromatography column(s) and/or chromatography membranes present in theMCCS or MCCS1 can include the same and/or different resins (e.g., any ofthe resins described herein or known in the art for use in recombinantprotein purification).

The two or more chromatography column(s) and/or chromatographic resin(s)present in the MCCS or MCCS1 can perform one or more unit operations(e.g., capturing a recombinant protein, purifying a recombinant protein,polishing a recombinant protein, inactivating viruses, adjusting theionic concentration and/or pH of a fluid including the recombinantprotein, or filtering a fluid including a recombinant protein). Innon-limiting examples, the MCCS or MCCS1 can perform the unit operationsof capturing a recombinant protein from a fluid (e.g., a liquid culturemedium) and inactivating viruses present in the fluid including therecombinant protein. The MCCS or MCCS1 can perform any combinations oftwo of more unit operations described herein or known in the art.

The chromatography column(s) and/or chromatographic membrane(s) presentin the MCCS or MCCS1 can be connected or moved with respect to eachother by a switching mechanism (e.g., a column-switching mechanism). TheMCCS or MCCS1 can also include one or more (e.g., two, three, four, orfive) pumps (e.g., automated, e.g., automated peristaltic pumps). Thecolumn-switching events can be triggered by the detection of a level ofrecombinant protein detected by UV absorbance corresponding to a certainlevel of recombinant protein in the fluid passing through the MCCS orMCCS1 (e.g., the input into and/or eluate from one or more of thechromatography column(s) and/or chromatographic membranes in the MCCS orMCCS1), a specific volume of liquid (e.g., buffer), or specific timeelapsed. Column switching generally means a mechanism by which at leasttwo different chromatography columns and/or chromatographic membranes inan MCCS or MCCS1 (e.g., two or more different chromatography columnsand/or chromatographic membranes present in the MCCS1 or MCCS2) areallowed to pass through a different step (e.g., equilibration, loading,eluting, or washing) at substantially the same time during at least partof the process.

The MCCS or MCCS1 can be a Periodic Counter-Current Chromatographysystem (PCCS). For example, the PCCS that is the MCCS or MCCS1 (i.e.,PCCS or PCCS1, respectively) can include four chromatography columns,where the first three columns perform the unit operation of capturing arecombinant protein from a fluid (e.g., a liquid culture medium), andthe fourth column of the PCCS performs the unit operation ofinactivating viruses in the fluid including the recombinant protein. APCCS that is the MCCS or MCCS1 can utilize a column-switching mechanism.The PCC system can utilize a modified ÄKTA system (GE Healthcare,Piscataway, N.J.) capable of running up to, e.g., four, five, six,seven, or eight columns, or more.

The MCCS or MCCS1 can be equipped with: one or more (e.g., two, three,four, five, six, seven, eight, nine, or ten) UV monitors, one or more(e.g., two, three, four, five, six, seven, eight, nine, or ten) valves,one or more (e.g., two, three, four, five, six, seven, eight, nine, orten) pH meters, and/or one or more (e.g., two, three, four, five, six,seven, eight, nine, or ten) conductivity meters. The MCCS or MCCS1 canalso be equipped with an operating system that utilizes software (e.g.,Unicorn-based software, GE Healthcare, Piscataway, N.J.) for sensingwhen a column-switching should occur (e.g., based upon UV absorbance,volume of liquid, or time elapsed) and affecting (triggering) thecolumn-switching events.

The MCCS or MCCS1 can further include one or more (e.g., two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty,twenty-one, twenty-two, twenty-three, or twenty-four) in-line bufferadjustment reservoir(s) and/or a buffer reservoir(s). In other examples,the MCCS or MCCS1 can include one or more (e.g., two, three, four, five,or six) break tanks that can hold fluid that cannot readily pass intoone or more of the chromatography columns and/or chromatographicmembranes in the MCCS or MCCS1. The systems described herein can includeone or more break tanks (e.g., a break tank described herein) in theMCCS, MCCS1, and/or MCCS2. Other examples of the systems describedherein do not include a break tank in the MCCS, MCCS1, or MCCS2, or donot include a break tank in the entire system. Other examples of thesystems described herein include a maximum of one, two, three, four, orfive break tank(s) (e.g., any break tank(s) described herein) in theentire system.

Second MCCS

The second MCCS (MCCS2) in the exemplary systems includes at least twochromatography columns, at least two chromatographic membranes, or atleast one chromatography column(s) and at least one chromatographicmembrane(s), and an outlet. The MCCS2 can any of the exemplary MCCSsdescribed herein, or can have one or more of any of the exemplaryfeatures of an MCCS (in any combination) described herein. Thechromatography column(s) and/or the chromatographic membrane(s) presentin the MCCS2 can have one or more of: any of the shapes, sizes, volumes(bed volumes), and/or unit operations described herein. Thechromatography column(s) and/or the chromatographic membrane(s) caninclude any of the exemplary resins described herein or known in theart. For example, the resin included in one or more of thechromatography column(s) and/or chromatographic membrane(s) present inthe MCCS2 can be a resin that utilizes a capture mechanism (e.g.,protein A-binding capture mechanism, protein G-binding capturemechanism, antibody- or antibody fragment-binding capture mechanism,substrate-binding capture mechanism, cofactor-binding capture mechanism,tag-binding capture mechanism, and/or aptamer-binding capturemechanism). Useful resins include, e.g., a cation exchange resin, ananion exchange resin, a molecular sieve resin, and a hydrophobicinteraction resin. Additional examples of resins are known in the art.The chromatography column(s) and/or chromatography membranes present inthe MCCS2 can include the same and/or different resins (e.g., any of theresins described herein or known in the art for use in recombinantprotein purification).

The chromatography column(s) and/or chromatographic membrane(s) presentin the MCCS2 can perform one or more unit operations (e.g., any of theunit operations described herein or any combination of the unitoperations described herein). In non-limiting examples, the MCCS2 canperform the unit operations of purifying a recombinant protein from afluid and polishing the recombinant protein present in the fluidincluding the recombinant protein. In other non-limiting examples, theMCCS2 can perform the unit operations of purifying a recombinant proteinpresent in a fluid, polishing a recombinant protein present in a fluid,and filtering a fluid including a recombinant protein. In anotherexample, the MCCS2 can perform the unit operations of purifying arecombinant protein present in a fluid, polishing a recombinant proteinpresent in a fluid, filtering a fluid including a recombinant protein,and adjusting the ionic concentration and/or pH of a fluid including arecombinant protein. The MCCS2 can perform any combination of two ofmore unit operations described herein or known in the art.

The chromatography column(s) and/or chromatographic membrane(s) presentin the MCCS2 can be connected or moved with respect to each other by aswitching mechanism (e.g., a column-switching mechanism). The MCCS2 canalso include one or more (e.g., two, three, four, or five) pumps (e.g.,automated, e.g., automated peristaltic pumps). The column-switchingevents can be triggered by the detection of a level of recombinantprotein detected by UV absorbance corresponding to a certain level ofrecombinant protein in the fluid passing through the MCCS2 (e.g., theinput into and/or eluate from one or more of the chromatographycolumn(s) and/or chromatographic membranes in the MCCS2), a specificvolume of liquid (e.g., buffer), or specific time elapsed.

The MCCS2 can be a Periodic Counter-Current Chromatography system (i.e.,PCCS2). For example, the PCCS2 can include three columns that performthe unit operation of purifying a recombinant protein from a fluid, anda chromatographic membrane that performs the unit operation of polishinga recombinant protein present in a fluid. For example, the three columnsthat perform the unit operation of purifying a recombinant protein froma fluid can include, e.g., a cationic exchange resin, and thechromatographic membrane that performs the unit operation of polishingcan include a cationic exchange resin. A PCCS2 can utilize acolumn-switching mechanism. The PCCS2 can utilize a modified ÄKTA system(GE Healthcare, Piscataway, N.J.) capable of running up to, e.g., four,five, six, seven, or eight columns, or more.

The MCCS2 can be equipped with: one or more (e.g., two, three, four,five, six, seven, eight, nine, or ten) UV monitors, one or more (e.g.,two, three, four, five, six, seven, eight, nine, or ten) valves, one ormore (e.g., two, three, four, five, six, seven, eight, nine, or ten) pHmeters, and/or one or more (e.g., two, three, four, five, six, seven,eight, nine, or ten) conductivity meters. The MCCS2 can also be equippedwith an operating system that utilizes software (e.g., Unicorn-basedsoftware, GE Healthcare, Piscataway, N.J.) for sensing when acolumn-switching event should occur (e.g., based upon UV absorbance,volume of liquid, or time elapsed) and affecting the column-switchingevents.

The MCCS2 can further include one or more (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one,twenty-two, twenty-three, or twenty-four) in-line buffer adjustmentreservoir(s) and/or a buffer reservoir(s). In other examples, the MCCS2can include one or more (e.g., two, three, four, five, or six) breaktanks (e.g., any of the break tanks described herein) that can holdfluid that cannot readily pass into one or more of the chromatographycolumns and/or chromatographic membranes in the MCCS2.

The MCCS2 includes an outlet through which the therapeutic protein drugsubstance can exit the system. The outlet can include, e.g., athreading, ribbing, or a seal that allows for a fluid conduit to beinserted or a vial designed to hold or store the purified recombinantprotein (e.g., therapeutic protein drug substance). An outlet caninclude a surface that can be used to seal a reduced bioburden vial orother such storage container onto the outlet in order to allow thepurified recombinant protein (e.g., therapeutic protein drug substance)to flow directly into the reduced bioburden vial or storage container.Non-limiting outlets that can be used in the present systems are knownand would be understood by those in the art.

The systems described herein can also include a fluid conduit that isdisposed between the MCCS1 and the MCCS2. Any of the fluid conduitsdescribed herein can be, e.g., a tube that is made of, e.g.,polyethylene, polycarbonate, or plastic. The fluid conduit disposedbetween the MCCS1 and the MCCS2 can further include one of more of thefollowing in any combination: one or more in-line buffer adjustmentreservoirs that are in fluid communication with the fluid conduit andare positioned such that the buffer stored within the in-line bufferadjustment reservoir(s) is added to the fluid present in the fluidconduit; a break tank (e.g., any of the break tank(s) described herein)that is in fluid communication with the fluid conduit and is positionedsuch that it can hold any excess fluid present in the fluid conduit thatis unable to readily feed into the MCCS2; and one or more filters thatare disposed in the fluid conduit such that they are capable offiltering (e.g., removing bacteria) the fluid present in the fluidconduit. Any of the in-line buffer adjustment reservoirs can include,e.g., a volume of between about 0.5 L to 50 L of buffer (e.g., at atemperature at or below 25° C., 15° C., or 10° C.).

The systems described herein can optionally include a fluid conduitdisposed between the final chromatography column or chromatographicmembrane in the MCCS2 and the outlet. The systems described herein canfurther include one or more filters in fluid connection with the fluidconduit disposed between the final chromatography column orchromatographic membrane in the MCCS2 and the outlet, such that thefilter can remove, e.g., precipitated material, particulate matter, orbacteria from the fluid present in the fluid conduit disposed betweenthe final chromatography column or chromatographic membrane in the MCCS2and the outlet.

Some examples of the systems provided herein also include a bioreactorthat is in fluid connectivity with the inlet of the MCCS or MCCS1. Anyof the exemplary bioreactors described herein or known in the art can beused in the present systems.

Some examples of the systems provided herein also include a pump system.A pump system can include one or more the following: one or more (e.g.,two, three, four, five, six, seven, eight, nine, or ten) pumps (e.g.,any of the pumps described herein or known in the art), one or more(e.g., two, three, four, or five) filters (e.g., any of the filtersdescribed herein or known in the art), one or more (e.g., two, three,four, five, six, seven, eight, nine, or ten) UV detectors, and one ormore (e.g., two, three, four, or five) break tanks (e.g., any of thebreak tanks described herein). Some examples of the systems providedherein further include a fluid conduit disposed between the pump and theinlet of the MCCS or MCCS1 (e.g., any of the exemplary fluid conduitsdescribed herein or known in the art). In some examples, this particularfluid conduit can include one or more (e.g., two, three, or four) pumps(e.g., any of the pumps described herein or known in the art) and/or oneor more (e.g., two, three, or four) break tanks (e.g., any of theexemplary break tanks described herein), where these pump(s) and/orbreak tank(s) are in fluid connection with the fluid present in thefluid conduit.

Some examples of the systems described herein further include a furtherfluid conduit connected to the fluid conduit between the pump and theinlet, where one end of the further fluid conduit is fluidly connectedto a bioreactor and the other end is fluidly connected to the fluidconduit between the pump and the inlet. This further fluid conduit caninclude a filter that is capable of removing cells from the liquidculture medium removed from the bioreactor (e.g., ATF cell retentionsystem).

The systems provided herein allow for the continuous production of apurified recombinant protein (e.g., therapeutic protein drug substance).As is known in the art, the systems can provide for the periodic elutionof a purified recombinant protein (e.g., therapeutic protein drugsubstance). The systems described herein can also result in a net yieldof purified recombinant protein (e.g., therapeutic protein drugsubstance) of at least about 5 g/day, at least about 10 g/day, at leastabout 15 g/day, at least about 20 g/day, at least about 30 g/day, or atleast about 40 g/day over a continuous period of at least about 5 days,at least about 10 days, at least about 15 days, at least about 20 days,at least about 25 days, at least about 30 days, at least about 40 days,at least about 50 days, at least about 60 days, at least about 70 days,at least about 80 days, at least about 90 days, or least about 100 days.

Methods of Reducing Bioburden of a Chromatography Resin that Include theUse of a Substantially Dry Chromatography Resin

Also provided herein are methods of reducing bioburden of achromatography resin that include (a) exposing a container comprising asubstantially dry chromatography resin to a dose of gamma-irradiationsufficient to reduce the bioburden of the container and thechromatography resin. Some embodiments of these methods further includeprior to step (a) drying a chromatography resin to remove liquid fromthe chromatography resin. Drying of a chromatography resin can beperformed using heat treatment (e.g., an oven) or a dessicator.Additional methods for drying a chromatography resin are known in theart.

Any of the conditions and doses for gamma-irradiation described hereincan be used in these methods. For example, the dose of gamma-irradiationcan be between about 15 kGy to about 45 kGy (e.g., between about 20 kGyto about 30 kGy). Any of the containers and chromatography resinsdescribed herein can be used in these methods. For example, thecontainer can be a storage vessel or a chromatography column. Thechromatography resin in these methods can include a protein ligand(e.g., protein A or protein G). In some examples, the chromatographyresin can include an anionic exchange chromatography resin (e.g., achromatography resin including N-benzyl-N-methyl-ethanolamine groups).In some examples, the chromatography resin is covalently attached to asurface of an article (e.g., a chip, membrane, or cassette). In someembodiments, the substantially dry chromatography resin does not containa significant amount of an antioxidant agent or a significant amount ofa chelator. Also provided are reduced bioburden chromatography resinsproduced by any of the methods described herein.

In some examples, the reduced bioburden chromatography resin producedhas a sterility assurance level (SAL) of between about 1×10⁻⁸ to about1×10⁻⁵ (e.g., a SAL of between about 1×10⁻⁷ to about 1×10⁻⁶). Thereduced chromatography resin produced can include at least one resinselected from the group consisting of: anionic exchange chromatographyresin, cationic exchange chromatography resin, affinity chromatographyresin, hydrophobic interaction chromatography resin, and size exclusionchromatography resin. In some examples, the reduced chromatography resinproduced includes an affinity chromatography resin comprising a proteinligand (e.g., protein A). In some examples, the reduced chromatographyresin produced includes an anionic exchange chromatography resin (e.g.,an anionic exchange chromatography resin includingN-benzyl-N-methyl-ethanolamine groups). Also provided are methods ofmaking a reduced bioburden packed chromatography column that includeproviding the reduced bioburden chromatography resin produced by any ofthe methods described herein; and packing the chromatography resin intoa reduced bioburden column in an aseptic environment. Also provided arereduced bioburden packed chromatography columns produced by any of themethods described herein.

Also provided are integrated, closed, and continuous processes forreduced bioburden manufacturing of a purified recombinant protein thatinclude: (a) providing a liquid culture medium including a recombinantprotein that is substantially free of cells; and (b) continuouslyfeeding the liquid culture medium into a multi-column chromatographysystem (MCCS) comprising at least one reduced bioburden packedchromatography column produced by any of the methods provided herein;where the process utilizes reduced bioburden buffer, is integrated, andruns continuously from the liquid culture medium to an eluate from theMCCS that is the purified recombinant protein. Also provided areintegrated, closed, and continuous processes for reduced bioburdenmanufacturing of a purified recombinant protein that include: (a)providing a liquid culture medium including a recombinant protein thatis substantially free of cells; (b) continuously feeding the liquidculture medium into a first multi-column chromatography system (MCCS1);(c) capturing the recombinant protein in the liquid culture medium usingthe MCCS1; (d) producing an eluate from the MCCS1 that includes therecombinant protein and continuously feeding the eluate into a secondmulti-column chromatography system (MCCS2); (e) continuously feeding therecombinant protein from the eluate into the MCCS2 and subsequentlyeluting the recombinant protein to thereby produce the purifiedrecombinant protein, where: the process utilizes reduced bioburdenbuffer, is integrated, and runs continuously from the liquid culturemedium to the purified recombinant protein, and at least one column inthe MCCS1 and/or MCCS2 contains a reduced bioburden packedchromatography column produced by any of the methods provided herein.Any of the exemplary aspects of integrated, closed, and continuousprocesses for reduced bioburden manufacturing of a purified recombinantprotein described herein can be used in these processes.

Methods of Producing Reduced Bioburden Membranes, Resins, CoatedMaterials, Chips, and Cassettes

Also provided herein are methods for producing a reduced bioburdenmembrane, resin, coated material, chip, or cassette that include:exposing a container including a composition including a membrane,resin, coated material, chip, or cassette (e.g., a cellulose-, agarose-,or a sugar-based membrane, resin, coated material, chip, or cassette);and at least one antioxidant agent and/or chelator to a dose ofgamma-irradiation sufficient to reduce the bioburden of the containerand the membrane, resin, coated material, chip, or cassette, where theat least one antioxidant agent and/or chelator is present in an amountsufficient to ameliorate the damage to the membrane, resin, coatedmaterial, chip, and cassette after exposure to the dose ofgamma-irradiation. In some examples, the cassette is a resin-containingcassette (e.g., any of the exemplary resins described herein or known inthe art). In some embodiments, the membrane, resin, coated material,chip, or cassette includes a protein A or protein G ligand covalentlyattached to at least one or part of its surface. In some embodiments,the composition includes a membrane, resin, coated material, chip, orcassette and a liquid including the at least one antioxidant agentand/or the at least one chelator. Any of the exemplary combinations andconcentrations of antioxidant agent(s) and/or chelator(s) describedherein can be used in any of these methods. Any of the exemplary liquidsdescribed herein can be used in any of these methods. In someembodiments, the composition is a dry material. Also provided herein isa reduced bioburden membrane, resin, coated material, chip, or cassetteproduced using any of the methods described herein. In some examples thecontainer is a sealed storage container or vessel.

Also provided herein are methods for producing a reduced bioburdenmembrane, resin, coated material, chip, and cassette that include:exposing a container including a substantially dry membrane, resin,coated material, chip, or cassette (e.g., a cellulose-, agarose-, or asugar-based membrane, resin, coated material, chip, or cassette) to adose of gamma-irradiation sufficient to reduce the bioburden of thecontainer and the membrane, resin, coated material, chip, or cassette.Some examples further include a step of drying the membrane, resin,coated materials, chip, or cassette prior to the exposing step. In someembodiments, the membrane, resin, coated material, chip, or cassetteincludes a protein A or protein G ligand covalently attached to itssurface. In some examples, the cassette is a resin-containing cassette(e.g., any of the exemplary resins described herein or known in theart). Also provided herein is a reduced bioburden membrane, resin,coated material, chip, or cassette produced using any of the methodsdescribed herein.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1 Effect of Gamma-Irradiation on Static BindingCapacity of Different Chromatography Resins

A set of experiments was performed to test the effect ofgamma-irradiation on the static binding capacity of three differentprotein A affinity chromatography resins: GE Mab Select SuRe™ (a highlycross-linked agarose resin having a particle size of 85 μm, and epoxyfunctional groups connecting protein A to the agarose), JSR LifeSciencesAmsphere ProA JWT203 (a porous poly-methacrylate resin having a particlesize of ˜50 μm, and epoxy functional groups connecting protein A to thepoly-methacrylate), and Kaneka KanCap A (a highly cross-linked cellulosehaving a particle size of 65-85 μm, with protein A linked to thecellulose through reductive amination). Each of the three resins wastreated with 15 kGy of gamma-irradiation, with the exception of MabSelect SuRe™ which was treated with the radiation dose of 25kGy, and acontrol sample of GE Mab Select SuRe™ resin was left untreated, loadedwith anti-TGFβ antibody (IgG4), and the level of the anti-TGFβ antibodyin the eluate was determined. Gamma-irradiation of each of the resins inthis example was performed in 50 mM phosphate, pH 7.0. The staticbinding curves of each of the gamma-irradiated resins and the untreatedcontrol resin are shown in FIG. 1. These data show thatgamma-irradiation reduces the binding capacity of each of the threetested protein A chromatography resins, with the JSR LifeSciencesAmsphere ProA JWT203 resin showing the smallest decrease in bindingcapacity in response to 15 kGy gamma-irradiation.

A next experiment was performed to determine whether gamma-irradiationof the JSR LifeSciences Amsphere ProA JWT203 resin or the GE Mab SelectSuRe™ resin results in a cleavage (and release) of protein A from thechromatography resin. In these experiments, JSR LifeSciences AmsphereProA JWT203 resin or the GE Mab Select SuRe™ resin were treated with 30kGy of gamma-irradiation, and then the level of protein A in thesupernatant was determined by measuring the absorbance at 280 nm. Thedata from this experiment show that little protein A is released fromeither of these two tested resins following treatment withgamma-irradiation (FIG. 2).

These data show that gamma-irradiation results in an immediate decreasein the binding capacity of affinity chromatography resin, and that suchloss in binding capacity is not due to cleavage of the protein ligandfrom the resin.

Example 2 Multiple of Gamma-Irradiation on Binding Capacity of ResinOver Multiple Cycles of Chromatography

A next set of experiments was performed to test the effect ofgamma-irradiation on the binding capacity of chromatography resin overmultiple cycles of chromatography. These experiments were performedusing JSR LifeSciences Amsphere ProA JWT203 resin as this resin showedthe least amount of loss of binding capacity in response togamma-irradiation in Example 1. Gamma-irradiation of each of the resinsin this example was performed in 50 mM phosphate, pH 7.0.

A first experiment was performed to test the effect of 15 kGygamma-irradiation on the ability of JSR LifeSciences Amsphere ProAJWT203 resin to bind to the anti-TGFβ antibody (IgG4) over multiplecycles of chromatography. An untreated JSR LifeSciences Amsphere ProAJWT203 resin was also used to perform multiple cycles of chromatography,as a positive control. At the end of each cycle, the resins were washedwith 0.1 N NaOH. The amount of the anti-TGFβ antibody in eluate fromeach cycle was determined. The data show that 15 kGy gamma-irradiationresults in an immediate, about 20% to 25%, decrease in binding capacityof the JSR LifeSciences Amsphere ProA JWT203 resin (i.e., in the firstcycle), but the binding capacity only decreases at a very slow rate overthe multiple additional cycles of chromatography (i.e., at a ratecomparable to the rate of decrease of binding capacity of the untreatedresin) (FIGS. 3, 6, and 7).

The minimal decrease in binding capacity of the 15 kGy gamma-irradiatedJSR LifeSciences Amsphere ProA JWT203 resin after the initial cycle ofchromatography is also apparent from a comparison of the chromatographsat cycle 7 and cycle 11 in these experiments (FIGS. 4 and 5). Thesefigures show that for both the untreated and the 15 kGy gamma-irradiatedJSR LifeSciences Amsphere ProA JWT203 resin there is only a modest, ifany, observable change in the binding capacity of these resins overmultiple cycles of chromatography.

Additional experiments were performed to test the change in bindingcapacity of JSR LifeSciences Amsphere ProA JWT203 resin left untreatedor treated with 29 kGy gamma-irradiation over multiple cycles ofchromatography. In these experiments, the resins were washed with 0.1 NNaOH at the end of each cycle, and the resins were loaded withanti-αβTCR monoclonal antibody (IgG1) at the start of each cycle. Theresulting data again show an about 20% to 25% initial drop in bindingcapacity of the 29 kGy gamma-irradiated resin as compared to theuntreated resin (FIG. 8). The binding capacity of the untreated and 29kGy gamma-irradiated resin only modestly decreased over multipleadditional cycles of chromatography (at about the same rate for theuntreated and 29 kGy gamma-irradiated resin) (FIG. 8).

A final set of experiments was performed to test the binding capacity ofuntreated, 15 kGy gamma-irradiated, and 29 kGy gamma-irradiated JSRLifeSciences Amsphere ProA JWT203 resin over multiple cycles ofchromatography. At the end of each cycle, the resins were washed with0.1 N NaOH, and the resin was loaded with either monoclonal anti-TGFβ(IgG4) antibody or monoclonal anti-αβTCR (IgG1) antibody (abTCR). Thedata show that irradiation with 15 kGy and 29 kGy of gamma-irradiationresults in substantially the same level of decrease in binding capacityof the JSR LifeSciences Amsphere ProA JWT203 resin, and that the levelof binding capacity of the 15 kGy and 29 kGy gamma-irradiated JSRLifeSciences Amsphere ProA JWT203 resin modestly decreases over multiplecycles of chromatography at the same rate as the untreated resin (FIG.9).

These data show that gamma-irradiation of a chromatography resin with aprotein ligand results in an immediate decrease in binding capacity, andthat the loss in binding capacity remains relatively stable overmultiple rounds of chromatography. These data suggest thatgamma-irradiation results in oxidative damage to chromatography resin(e.g., chromatography resin containing a protein or peptide ligand, suchas affinity chromatography resin) during gamma-irradiation, and thatloss in binding capacity of chromatography resin (e.g., chromatographyresin containing a protein or peptide ligand, such as affinitychromatography resin) during gamma-irradiation can be prevented,ameliorated, or reduced by gamma-irradiating the chromatography resin inthe presence of at least one chelator and/or antioxidant agent.

Example 3 Effect of Gamma-Irradiation on Binding Capacity on GE MabSelect SuRe™ Resin

A set of experiments was performed to determine the effect ofgamma-irradiation on the binding capacity of another protein ligandchromatography resin (GE Mab Select SuRe™ resin) over multiple cycles ofchromatography. The resin was exchanged into 50 mM sodium phosphate, pH7.0, and then treated with 29 kGy gamma-irradiation in a 30-mL Nalgenebottle. The resin was then packed into a 0.66-cm diameter column with abed height of 3 cm (1 mL column). This column was then used to capture amonoclonal IgG1 antibody in a cell culture medium over multiple cyclesof chromatography. An untreated GE Mab Select SuRe™ resin was also usedto perform multiple cycles of chromatography as a positive control. Atthe end of each cycle, the resins were washed with 0.1 N NaOH. Theamount of IgG1 in eluate from each cycle was determined. The data showthat 29 kGy gamma-irradiation results in an immediate, about 25% toabout 30%, decrease in the binding capacity of the GE Mab Select SuRe™resin (i.e., in the first cycle) (FIGS. 11 and 12). After the firstcycle of chromatography, the 29 kGy gamma-irradiated GE Mab Select SuRe™resin only showed a very slow rate of decrease in binding capacity overmultiple additional cycles of chromatography (i.e., at a rate comparableto the rate of decrease of binding capacity of the untreated resin overmultiple chromatography cycles) (FIGS. 11 and 12). These data aresimilar to the data obtained for the JSR Protein A resin described inExample 2 and the mechanism for loss of resin binding capacity due togamma-irradiation appears to be similar.

Example 4 Effect of Gamma-Irradiation on Static Binding Capacity of MAbSelect SuRe™ LX Resin

The data described in Example 1 show that gamma-irradiation reduces thestatic binding capacity of three different Protein A chromatographyresins (GE Mab Select SuRe™, JSR LifeSciences Amsphere™ ProA JWT203, andKaneka KanCapA™). Gamma-irradiation was tested with another protein Achromatography resin: GE Mab Select SuRe™ LX. GE Mab Select SuRe™ LXresin has an agarose backbone and a similar protein A ligand as MabSelect SuRe™ resin, but has a higher ligand density as compared to MabSelect SuRe™ resin. The resin buffer was exchanged from 20% ethanol to50 mM sodium phosphate, pH 6.0, and was then subjected to 29-kGygamma-irradiation. The static binding of the resin was determined asdescribed in Example 1 (with static binding capacity of the resindetermined before and after gamma-irradiation). The data indicate asimilar drop in static binding capacity of the resin aftergamma-irradiation (FIG. 13). This gamma-irradiation-induced decrease instatic binding capacity of the GE Mab Select SuRe™ LX resin is similarto the drop in static binding capacity observed for the three protein Achromatography resins tested in Example 1 following gamma-irradiation(FIG. 1). The data in FIG. 13 show that gamma-irradiation of Mab SelectSuRe™ LX results in an immediate decrease in the static binding capacityof the resin.

Example 5 Reduction of the Gamma-Irradiation-Induced Decrease in BindingCapacity of a Chromatography Resin

The data in the previous examples show that gamma-irradiation ofchromatography resin in a dose that reduces the bioburden of the resin,also results in a drop in the binding capacity of the resin: a decreasein the resin binding capacity of about 20% to about 40% as compared tothe binding capacity of a control untreated resin. The extent of thedecrease in binding capacity caused by gamma-irradiation does not appearto depend on the protein A ligand attached to the resin and/or the beadchemistry of the resin. This additional set of experiments was performedto identify buffered solutions that would prevent the loss in bindingcapacity of a chromatography resin that occurs during gamma-irradiation.GE Mab Select SuRe™ LX was used as the chromatography resin in theseexperiments. GE Mab Select SuRe™ LX has protein A covalently immobilizedonto the porous matrix of each bead. The resin was first bufferexchanged from 20% ethanol to a different buffer including one or moreantioxidant agents or a control buffer not including any antioxidantagents. The tested buffered solutions are shown in Table 1 below. In acontrol experiment, the resin was gamma-irradiated as a dry compositionin the absence of antioxidant agents. These resin samples were dried inan oven at 23° C. for 2 days such that water content was minimizedduring gamma-irradiation. Each resin sample shown in Table 1 wasgamma-irradiated at a dose of 29 kGy in a 30-mL Nalgene bottle. Thestatic binding capacity was determined for the gamma-irradiated resinsamples and for the untreated (non-irradiated) resin samples using themethods described in Example 1.

TABLE 1 Tested GE Mab Select SuRe ™ LX Resin Samples and BufferedSolutions Resin Base buffer Quenchers Virgin MabSelect 50 mM Sodium NoneSuRe LX phosphate, pH 6.0 29 kGy MabSelect 50 mM Sodium None SuRe LXphosphate, pH 6.0 29 kGy MabSelect 50 mM Sodium 100 mM Mannitol SuRe LXphosphate, pH 6.0 29 kGy MabSelect 50 mM Sodium 100 mM Methionine SuReLX phosphate, pH 6.0 29 kGy MabSelect 50 mM Sodium 100 mM sodiumascorbate SuRe LX phosphate, pH 6.0 29 kGy MabSelect 50 mM Sodium 100 mMHistidine SuRe LX phosphate, pH 6.0 29 kGy MabSelect 50 mM Sodium 50 mMMethionine + SuRe LX phosphate, pH 6.0 50 mM Histidine 29 kGy MabSelect50 mM Sodium 33 mM Methionine + SuRe LX phosphate, pH 6.0 33 mMHistidine + 33 mM sodium ascorbate 29 kGy MabSelect 50 mM Sodium 25 mMsodium ascorbate + SuRe LX phosphate, pH 6.0 25 mM Methionine + 25 mMMannitol + 25 mM Histidine Dried Virgin None None MabSelect SuRe LXDried 29 kGy None None MabSelect SuRe LX

The data show that gamma-irradiation of the chromatography resin in thepresence of one or more antioxidant agents decreased the loss in bindingcapacity caused by gamma-irradiation (FIG. 14 and Table 2). The bufferof 25 mM sodium ascorbate, 25 mM methionine, 25 mM mannitol, 25 mMhistidine, 50 mM sodium phosphate, pH 6.0 (referred to hereafter as“SMMH” buffer) completely mitigated the gamma-irradiation-induced lossin binding capacity of the resin (i.e., there was no loss in bindingcapacity observed between the untreated control resin and the resingamma-irradiated with a dose of 29 kGy in the presence of SMMH buffer)(FIG. 14 and Table 2). The 29-kGy gamma-irradiated dried resin samplehad the same binding capacity as the untreated resin sample. These dataindicate that gamma-irradiating a dry chromatography resin is anotherway to decrease the extent of gamma-irradiation-induced loss in resinbinding capacity.

TABLE 2 Specific Binding Capacity for Tested GE Mab Select SuRe ™ LXResin Samples SBC (mg/ Resin Base buffer Quenchers mL) Virgin MabSelect50 mM Sodium None 50 SuRe phosphate, pH 7.0 29 kGy MabSelect 50 mMSodium None 32.8 SuRe phosphate, pH 7.0 Virgin MabSelect 50 mM SodiumNone 70 SuRe LX phosphate, pH 6.0 29 kGy MabSelect 50 mM Sodium None 53SuRe LX phosphate, pH 6.0 29 kGy MabSelect 50 mM Sodium 100 mM Mannitol57 SuRe LX phosphate, pH 6.0 29 kGy MabSelect 50 mM Sodium 100 mMMethionine 64.5 SuRe LX phosphate, pH 6.0 29 kGy MabSelect 50 mM Sodium100 mM sodium 66 SuRe LX phosphate, pH 6.0 ascorbate 29 kGy MabSelect 50mM Sodium 100 mM Histidine 69 SuRe LX phosphate, pH 6.0 29 kGy MabSelect50 mM Sodium 50 mM Methionine + 63.5 SuRe LX phosphate, pH 6.0 50 mMHistidine 29 kGy MabSelect 50 mM Sodium 33 mM Methionine + 69 SuRe LXphosphate, pH 6.0 33 mM Histidine + 33 mM sodium ascorbate 29 kGyMabSelect 50 mM Sodium 25 mM sodium 74 SuRe LX phosphate, pH 6.0ascorbate + 25 mM Methionine + 25 mM Mannitol + 25 mM Histidine DriedVirgin None None 75 MabSelect SuRe LX Dried 29 kGy None None 74MabSelect SuRe LX

A further experiment was performed to test the performance of resingamma-irradiated with a dose of 29 kGy in the presence of the SMMHbuffer and untreated resin over multiple chromatography cycles. Eachtested resin was packed into a 0.66-cm diameter and 3-cm height column.The columns were loaded with cell culture medium containing a monoclonalIgG1 antibody. As observed in the static binding capacity experiments(FIG. 14), the dynamic binding capacity was comparable between theuntreated resin and the resin gamma-irradiated with a dose of 29 kGy inthe presence of the SMMH buffer for all cycles (FIGS. 15 and 16). Inaddition, other critical quality and process performance indicators werealso comparable: the percentage of high molecular weight speciesmeasured by size exclusion chromatography-high performance liquidchromatography (SEC-HPLC) and the concentration of host cell protein(HCP) in the eluate (Table 3). FIG. 17 is a non-denaturing sodiumdodecyl sulfate polyacrylamide gel depicting the comparable purity ofthe eluate over multiple cycles from the untreated resin and the resingamma-irradiated at a dose of 29 kGy in the presence of the SMMH buffer.

TABLE 3 Percentage of High Molecular Weight Species and Host CellProtein in Eluate of Untreated GE Mab Select SuRe ™ LX Resin and GE MabSelect SuRe ™ LX Resin Gamma-Irradiated in the Presence of SMMH BufferProtein A % HCP Protein A % HCP eluate HMWS (ng/mg) eluate HMWS (ng/mg)Virgin eluate 1.55 173 SMMH eluate 1.82 107.6 cycle 1 cycle 1 Virgineluate 1.73 247.2 SMMH eluate 1.75 N/A cycle 3 cycle 2 Virgin eluate1.89 289.5 SMMH eluate 1.59 146.2 cycle 5 cycle 3 The percentage highmolecular weight species was measured by size exclusion chromatographyhigh performance liquid chromatography (SEC-HPLC) using a Tosoh G3000SWx1 column and the host cell protein concentration was measured usingan ELISA assay.

Additional buffered solutions were tested for their ability to reducegamma-irradiation-induced loss in binding capacity of chromatographyresin. In these experiments, a single chromatography cycle was performedto determine binding capacity in dynamic mode. For each tested buffer, acolumn similar to the one described above (0.66-cm diameter×3-cm height)was packed and loaded with cell culture medium containing a monoclonalIgG1. As observed in the static binding capacity experiments (FIG. 14),each buffer containing at least one antioxidant agent reduced the lossin resin binding capacity caused by gamma-irradiation (Table 4). Thesedata show that the presence of at least one antioxidant agent can reducethe loss in resin binding capacity caused by gamma-irradiation. Inaddition, these data also suggest that chelators (which bind and preventredox active metals from generating reactive oxygen and nitrogenspecies) would also be able to decrease the loss in resin bindingcapacity caused by gamma-irradiation.

TABLE 4 Data from Single Chromatography Cycle on GE MabSelect SuRe LXResin (Untreated and 29 kGy-Irradiated in the Presence or Absence ofQuenchers) with Cell Culture Harvest Containing IgG1 Protein in BoundResin condition Eluate (mg) protein (mg) Virgin MabSelect SuRe LX 70 6829 kGy MabSelect SuRe LX 69 67 with SMMH 29 kGy MabSelect SuRe LX 68 67with 100 mM Histidine 29 kGy MabSelect SuRe LX 64 63 with 100 mMMethionine 29 kGy MabSelect SuRe LX 62 61 with 100 mM SA 29 kGyMabSelect SuRe LX 51 51

Overall, these data suggest that gamma-irradiation of chromatographyresin in the presence of at least one antioxidant agent and/or at leastone chelator can decrease the loss in resin binding capacity caused bygamma-irradiation. The data also suggest that the presence of at leastone antioxidant agent and/or at least one chelator protect theimmobilized protein A ligand, as well as the resin base matrix, fromdamage caused by free radicals formed during gamma-irradiation. Thepresence of at least one antioxidant agent and/or at least one chelatorcan also be used in other bioprocessing applications that include thegamma-irradiation of a material at a dosage that may cause damage to amatrix and/or functional groups. For example, compositions containing atleast one antioxidant agent and/or at least one chelator agent can beused to prevent gamma-irradiation induced damage to cellulose membranesand other cellulose-, agarose-, or sugar-based resins, membranes, ormaterials.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of reducing bioburden of achromatography resin comprising: exposing a container comprising acomposition comprising (i) a chromatography resin and (ii) a liquidcomprising at least one chelator to a dose of gamma-irradiationsufficient to reduce the bioburden of the container and thechromatography resin, wherein the at least one chelator is present in anamount sufficient to ameliorate the loss of binding capacity of thechromatography resin after exposure to the dose of gamma-irradiation. 2.The method of claim 1, wherein the container is a storage vessel.
 3. Themethod of claim 1, wherein the container is a chromatography column. 4.The method of claim 1, wherein the container is a packed chromatographycolumn.
 5. The method of claim 1, wherein the composition is a slurry ofsedimented chromatography resin in the liquid.
 6. The method of claim 5,wherein the liquid is a buffered solution.
 7. The method of claim 1,wherein the at least one chelator is selected from the group consistingof: ethylenediaminetetraacetic acid (EDTA),2,3-dimercapto-1-propanesulfonic acid sodium (DMPS), dimercaptosuccinicacid (DMSA), metallothionin, and desferroxamine.
 8. The method of claim1, wherein the chromatography resin is selected from the groupconsisting of: an anion exchange chromatography resin, a cation exchangechromatography resin, an affinity chromatography resin, a hydrophobicinteraction chromatography resin, and a size exclusion chromatographyresin.
 9. The method of claim 1, wherein the dose is between about 15kGy and about 45 kGy.
 10. A method of reducing bioburden of achromatography resin comprising: exposing a container comprising acomposition comprising a chromatography resin and at least oneantioxidant agent to a dose of gamma-irradiation sufficient to reducethe bioburden of the container and the chromatography resin, wherein theat least one antioxidant agent is present in an amount sufficient toameliorate the loss of binding capacity of the chromatography resinafter exposure to the dose of gamma-irradiation.
 11. The method of claim10, wherein the container is a storage vessel.
 12. The method of claim10, wherein the container is a chromatography column.
 13. The method ofclaim 10, wherein the container is a packed chromatography column. 14.The method of claim 10, wherein the composition further comprises atleast one chelator.
 15. The method of claim 10, wherein the compositionis a slurry of sedimented chromatography resin in a liquid comprisingthe at least one antioxidant agent.
 16. The method of claim 10, whereinthe composition is a solid mixture.
 17. The method of claim 10, whereinthe composition comprises at least one antioxidant agent selected fromthe group consisting of: reduced glutathione, reduced thioredoxin,reduced cysteine, a carotenoid, melatonin, lycopene, tocopherol, reducedubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, a flavonoid, aphenolpropanoid acid, lidocaine, naringenin, fullerene, glucose,histidine, methionine, mannitol,4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, and dimethylmethoxychromanol.
 18. The method of claim 15, wherein the liquid is a bufferedsolution.
 19. The method of claim 10, wherein the chromatography resinis selected from the group consisting of: an anion exchangechromatography resin, a cation exchange chromatography resin, anaffinity chromatography resin, a hydrophobic interaction chromatographyresin, and a size exclusion chromatography resin.
 20. The method ofclaim 10, wherein the dose is between about 15 kGy and about 45 kGy. 21.A method of reducing bioburden of a chromatography resin comprising:exposing a container comprising a composition comprising achromatography resin and methionine and/or histidine to a dose ofgamma-irradiation sufficient to reduce the bioburden of the containerand the chromatography resin, wherein the methionine and/or histidine ispresent in an amount sufficient to ameliorate the loss of bindingcapacity of the chromatography resin after exposure to the dose ofgamma-irradiation.
 22. The method of claim 21, wherein the compositioncomprises a chromatography resin and between 5 mM and about 150 mMmethionine.
 23. The method of claim 21, wherein the compositioncomprises a chromatography resin and between about 5 mM and about 150 mMhistidine.
 24. The method of claim 21, wherein the composition comprisesa chromatography resin, between about 5 mM and about 150 mM methionine,and between about 5 mM and about 150 mM histidine.
 25. The method ofclaim 21, wherein the container is a storage vessel or a chromatographycolumn.
 26. The method of claim 21, wherein the container is a packedchromatography column.
 27. The method of claim 21, wherein thecomposition is a solid mixture or a slurry of sedimented chromatographyresin in a liquid comprising the methionine and/or histidine.
 28. Themethod of claim 27, wherein the liquid is a buffered solution.
 29. Themethod of claim 21, wherein the chromatography resin is selected fromthe group consisting of: an anion exchange chromatography resin, acation exchange chromatography resin, an affinity chromatography resin,a hydrophobic interaction chromatography resin, and a size exclusionchromatography resin.
 30. The method of claim 21, wherein the dose isbetween about 15 kGy and about 45 kGy.